|Other Abstract||Two major lipophilic compounds, 2-methylisoborneol (2-MIB) and geosmin, are primarily responsible for imparting earthy-musty off-flavors in aquaculture and drinking water industries. Cyanobacteria have been attributed to the main biological sources of the two odorous compounds in eutrophic aquatic systems. As China is a developing country, the off-flavor problem will be an austere aquatic environmental problem for the future, due to environmental protection lagging behind the rapid economic growth. Thus making a study of cyanobacteria-producing off-flavors will provide insight into scientific significance and attempt to meet the urgent demands to our country. Based on the above reasons, the present dissertation established an instrumental method for the analysis of off-flavors in fish, investigated annual dynamics and biological origins of earthy-musty odor in Xionghe Reservoir, and studied the physiological characteristics and odorous compounds production in Pseudanabaena sp. and Lyngbya kuetzingii. The main results are summarized as follows:
1) 2-MIB and geosmin were determined by microwave mediated distillation with headspace solid-phase microextraction and gas chromatography-mass spectrum (MWDE-SPME-GC-MS). It was investigated and discussed the effects of the key parameters, microwave processing time and carrier gas flow rate, in microwave mediated distillation on extracts. It’s concluded that both 6 min of microwave time and 70 mL•min-1 of nitrogen flow were the optimized condition. On the optimized condition, odorous compounds could be completely distilled from fish flesh. Then the odorous compounds were absorbed in the fiber under HS-SPME. Finally, they were desorbed at 250 °C and determined by GC-MS. The limits of detection for 2-MIB and geosmin in fish were both 0.1 µg•kg-1. There were good linear correlation for the two odorous compounds in the range of 1~20 µg•kg-1, and the calibration coefficients were 0.987 and 0.995, respectively. Therefore, trace levels of off-flavor at ppb in fish could be quantified by this method with satisfactory result.
2) To find out the doubted odorous compounds and their origins, the annual variation of physicochemical parameters, algae and odorous compounds at three sites in Xionghe Reservoir were investigated. During May 2007 to Apr 2008, the algal composition, algal cell number, and earthy-musty odorous compounds were identified monthly and monitored in Site A, B and C. Meanwhile, physicochemical parameters such as total nitrogen (TN), total phosphorus (TP), dissolved oxygen (DO), pH, transparency, water temperature and chlorophyll a (chl a) were determined. Two earthy-musty odorous compounds, 2-MIB and geosmin existed in the reservoir were identified by using GC-MS. During the episodes, a great deal of Anabaena circinalis was present on the surface of water body. The highest concentration of geosmin reached 2.7 µg•L-1 while no 2-MIB was detected in July 2007, which showed that geosmin was mainly responsible for the off-flavor episodes in summer. The biological origin of 2-MIB may be related to Pseudanabaena sp., a MIB-producing cyanobacteria, isolated from sediment in Xionghe Reservoir. The concentrations of 2-MIB and geosmin were significantly correlated with the biomass of Pseudanabaena sp. and Anabaena circinalis (P < 0.01), respectively.
3) A filamentous, nonheterocystous cyanobacterium was isolated from Xionghe Reservoir with Pasteur-type pipette and a series of purification. Based on phenotypic analysis and molecular phylogenetic analyses dealt with the sequence of the 16S rRNA gene, this strain was named Pseudanabaena sp., which could produce 2-MIB detected by GC-MS.
4) It was investigated that the effect of culture time on the growth, extracellular 2-MIB production and production rate, and the relationship between extracellular 2-MIB production and cell numbers. The results showed that the specific growth rate (µ) of Pseudanabaena sp. was up to 0.255 ± 0.011 d-1. Extracellular 2-MIB production was increasing during the lag, exponential and late-stationary phase. Extracellular 2-MIB production rate (2-MIB production per unit cell number) was increasing during the lag, late-stationary and decline phase, while decreased at exponential phase. There was a significantly positive correlation between extracellular 2-MIB production and cell numbers.
5) The cell number, 2-MIB production and production rate of Pseudanabaena sp. response to temperature were compared with that response to light intensity. Pseudanabaena sp. was more sensitive to light intensity than to temperature. 25 μmol•m-2•s-1 was the optimum light intensity to the growth, while Pseudanabaena sp. could grow well among 20、25 and 30 °C. 2-MIB production increased as temperature increasing, and reached the peak value at 35 °C. Both high and low light intensities could decrease the 2-MIB production, and the peak value was only observed at 40 μmol•m-2•s-1. Increasing light intensity could stimulate the release of 2-MIB into culture medium, while temperature did not play a part in the release. However, the variety of 2-MIB production rate under different temperatures was the same as that at different light intensities. 2-MIB production rate was negatively correlated with cell number.
6) The characteristics of the growth, geosmin production and production rate of L. kuetzingii were studied in the laboratory. The specific growth rate (µ) of L. kuetzingii was only 0.130 ± 0.008 d-1. Dissolved geosmin production increased as the growth progressed, when reached its maximum value, and then declined. Geosmin production rate (geosmin production per unit chl a concentration) decreased rapidly at the exponential phase, while small amplitude was occurred at late-stationary phase. The ratio of intracellular dissolved geosmin to total geosmin was relatively high at exponential phase. Otherwise, the ratio of extracellular geosmin was high at late-stationary phase.
7) The effects of light intensity, temperature, phosphate-P, nitrate-N, and the micronutrients calcium and copper on the growth, geosmin production and production rate by L. kuetzingii were determined. Of the 3 temperatures—10, 25, and 35 °C—tested, 10 °C yielded the maximal geosmin production and production rate, but optimal chl a production was observed at 25 °C. In the studies on light intensity, the maximal geosmin production and producton rate were observed at 10 µmol•m–2•s–1, while 20 µmol•m–2•s–1 yielded the optimal chl a production. Chl a synthesis by L. kuetzingii was positively correlated to PO4-P and NO3-N concentrations. The production and production rateof geosmin were remarkably promoted by 0 and 0.2 mg PO4-P•L-1, whereas 0 and 2.47 mg NO3-N•L-1 maximized the production rate with a significant difference (P < 0.01). Geosmin production increased with an increase in the Ca2+ concentration in the culture medium, while chl a synthesis was inhibited at 0.980 g Ca2+•L-1. It was suggested that more geosmin was synthesized with lower chl a demand. The Cu2+ toxicity threshold of L. kuetzingii was 0.4 mg•L-1. Chl a and geosmin were not detected when the Cu2+ concentration in the growth media was higher than 0.4 mg Cu2+•L-1. Chl a and geosmin production were negatively correlated with Cu2+ ion concentrations lower than 0.4 mg Cu2+•L-1 (R = –0.974, P < 0.01; and R = –0.860, P < 0.05, respectively). Meanwhile, the relative amounts of extra- and intracellular dissolved 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 dissolved geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under unfavorable growth conditions (e.g., 10 °C, 10 µmol•m–2•s–1, 0 & 0.2 mg PO4-P•L-1, 0 & 2.47 mg NO3-N•L-1, 0.980 g Ca2+•L-1), less geosmin was released into the culture medium, and mainly accumulated in cells.|