Characterization of Odorous Compoundsin Rotten Blue-green Algae

Transcription

1 Agric. Biol Chern., 46 (7), , Characterization of Odorous Compoundsin Rotten Blue-green Algae Akio Yasuhara Keiichiro Division of Chemistry Physics, National Institute for Environmental Studies, Yatabe, Tsukuba-gun, Ibaraki 305, Japan Received October 30, 1981 Fuwa Blue-green algae, commonly called "Aoko," were taken from Lake Kasumigaura in Japan the components which contributed to the rotten odor of the algae were analyzed by gas chromatography computerized gas chromatography-mass spectrometry. Vacuumdistillation of the algae in a frozed state was the best method for effectively isolating volatiles odorous componentswithout changes. Large amounts of alcohols, fatty acids esters were detected in rotten algae in contrast with fresh algae. The mammalodorouscomponentin rotten algae was butanoic acid. Other odorous compounds were butanol, 3-methylbutanol, 3-methylbutanoic acid, hexanoic acid, butyl butanoate butyl hexanoate. Recently most freshwater lakes have become polluted by waste water from industrial civic activities. A number of blue-green algae grow actively in eutrophic lakes from spring to autumn causing damage to fish stocks. Moreover, strong malodors evolve from decomposition of too muchblue-green algae cause people in the surrounding region psychological harm. Fresh blue-green algae have an odor similar to that of water grasses, while rotten blue-green algae have a nauseating odor resembling rancid butter. It is not known which compounds contribute predominantly to the malodor. Studies of rotten odors are seldom carried out because of local pollution absence of physical injuries. Kikuchi et al.x) detected geosmin in a culture of algae isolated from Lake Biwa concluded that geosminwas a major cause of the malodor of algae. It is well knownthat geosmin is one of the metabolites in actinomycetes,2) but other investigators have not detected it in algae. Probably they detected geosmin which was formed by actinomycetes attached to the algae. There is a distinct difference between the odor of rotten blue-green algae the musty odor of geosmin. 3- Methylbutanoic acid, hexanal heptanal were also identified in diatoms isolated from Lake Biwa by Kikuchi et al.3) Jenkins et al.4) detected manysulfur-containing compounds by both spot tests gas chromatography, when blue-green algae were cultivated. There are several problems in these studies, which cannot be easily solved. Firstly, exact classification of blue-green algae cannot be made. Secondly, artificial cultivation of bluegreen algae is very difficult. Thirdly, estimation of microbial effects caused by many kinds of bacteria attached to algae is almost impossible. It is also not clear how decomposition ofbluegreen algae is affected by environmental changes. In this study, a sample of blue-green algae, commonly called "Aoko," obtained from Lake Kasumigaura, was used instead of artificially cultivated blue-green algae. Isolation identification of odorous compounds from the rotten blue-green algae was accomplished. MATERIALS AND METHODS Sampling extraction procedures. Blue-green algae were collected with a Plankton net from Lake Kasumigaura in Ibaraki Prefecture, Japan, on August 13, 1980, packed into three bottles. The algae could not

2 1762 A. Yasuhara K. Fuwa be separated completely from the water. One part of the algae (sample No. 1, wet volume 1.5 liters, dry weight 17.27g) was frozen rapidly then vacuum-distilled under a pressure of ca. 10~4 Torr at room temperature. The distillate was shaken with ethyl ether (700ml) after the addition of sodium chloride. The extract was dried over anhydrous sodium sulfate concentrated to a volume of 2.5ml with a Kuderna-Danish concentrator under atmospheric pressure. The second part of the algae (sample No. 2, wet volume 2.05 liters, dry weight 24.66g) was stored for a week at 10 C then for 4 days at room temperature (20~24 C). The rotten suspension was rapidly frozen vacuum-distilled. Treatment of the distillate was the same as described above for sample No. 1. The volume of the concentrated extract was 7.2 ml. The third part of the algae (sample No. 3, wet volume 1.7 liters, dry weight 22.36g) was stored for 3 weeks at 10 C. The decomposed suspension of the algae was rapidly frozen vacuum-distilled. The distillate was treated as described above for sample No. 1. The extract was concentrated to a volume of2.4 ml. Each concentrated extract was analyzed by gas chromatography (GC) gas chromatography-mass spectrometry (GC/MS). Mass spectrometry. Massspectra were measuredwith a JEOL Model JMS-D100 mass spectrometer connected to a 2000 JEOL mass JGC-20K data analysis gas chromatograph system. The gas-chromatographic a JEOL JMAconditions column of a were different the sameas size described (3m x 2mmi.d.) above, except was used that a helium gas was used as carrier gas. The separator temperaturditions for were GC/MSwas as follows: 250 C. ionizing The mass-spectrometric current, 3 x 10~4A; ionizing energy, 75 ev; accelerating voltage, 3 kv; scan range, con- m/z sec. 10~400; scan speed, 2.6 sec/scan; scan interval, 5 Sensory test. Effluent gas from the column outlet of the gas stream chromatograph was channeled was to divided the detector into in two the streams. gas chromatograph One Pleasantness or the unpleasantness, other stream was strongness smelled or by weakness, a man. lightness or heaviness were considered, although the recognition or description was relatively subjective. RESULTSANDDISCUSSION odorous Four methods components were of applied fresh for blue-green isolation algae of in with preliminary a Nickerson-Likens experiments. apparatus Steam distillation successfully used at first, because the was odor un- altered largely by thermal decomposition. was Generally on heating, odorous so heating components must be change avoided easily as much as possible. Next, liquid-liquid extractionique, was since tried the formation found to of be an a emulsion poor tech- lots of nonvolatile compoundsin the extract Gas chromatography. Gas chromatograms were obtained with a Shimadzu Model GC-7A gas chromatograph equipped with flame ionization detectors. The glass column (3m x 3 mm i.d.) was packed with 5% Thermon-3000 (Chromosorb W-AW-DMCS, mesh) which was obtained from Shinwa Kako Co., Ltd. The column temperature was set at 50 C for 2 min, followed by an increase to 210 C at a rate of4 C/min, then held at 210 C until completion of analysis. The injection temperature was 250 C the nitrogen carrier gas flow rate was 40ml/ min. Quantification was carried out using peak heights all values were corrected with recovery coefficients Time(min) Fie. 1. Gas Chromatogram of the Volatile Components in Fresh Blue-green Algae (Sample No. 1).

3 S3hijlJukF ICO 8 Odorous Compoundsin Rotten Blue-green Algae 1763.j<3»U Time (min ) Fig. 2. Gas Chromatogram of the Volatile Components in Rotten Blue-green Algae (Sample No. 2). felj ^uljuu ^y Time(min) Fig. 3. Gas Chromatogramof the Volatile Components in Rotten Blue-green Algae (Sample No. 3). interferred with the extraction the gaschromatographic analysis, respectively. Continuous extraction using a soxhlet extractor tried as a third method also had the same faults. Vacuumdistillation in a frozen state was the best technique at the time, though it was time-consuming. The gas chromatograms are shown in Figs. 1, 2 3. The results of identification are shown in Table I. Components identified by GC/MSwere also confirmed by peak enhancement on co-injection of the sample an authentic compound into the gas chromatograph. Somecompoundswere tentatively identified only by GC/MS. Amounts of volatiles differed largely between fresh rotten algae. It was concluded that large amounts of volatiles were formed by microbial decomposition. Concentrations of volatile compounds in fresh rotten algae are shown in Table II with the results of recovery tests. All recovery coefficients were satisfactory. It is not clear whether aliphatic hydrocarbons were present originally or not, as Apiezon grease was used for sealing in the vacuum distillation. These aliphatic hydrocarbons were partially detected in a blank test. In all cases, however, aliphatic hydrocarbons are not an important cause of the bad odor. Quantities of carbonyl compounds, which were expected to contribute to the malodor, were very small variable, so they were found not to be an important cause of the malodor, see Table II. Fenchone was detected from fresh algae in another experiment which was not described in the experimental section. Most alcohols had a very unpleasant pungent odor like fusel oil. So they were important contributors to the odor of rotten algae. It is very interesting that phenethyl alcohol was detected in both fresh rotten

4 1764 A. Yasuhara K. Fuwa Table I. Identified Compounds in Fresh Rotten Blue-green Algae Peak _, Retention time X T Compound... No. ^ (mm) 1 Propanol Ethyl butanoate Butyl acetate Methylpropanol Di-( l -methylethyl) ether* Butanol l-penten-3-ol Methylpropyl butanoate Methyl- l -butanol Butyl butanoate ,3-Butanedione Ethyl hexanoate Methylbutyl butanoate Pentyl butanoate Cyclopentanol* Hexanol Methylpropyl hexanoate Butyl hexanoate Acetic acid Methylbutyl hexanoate Propanoic acid Methylbutyl octanoate Methylpropanoic acid Butanoic acid Hexadecane Methylbutanoic acid Heptadecane Pentanoic acid Methylheptadecane Fenchone Branched octadecane Methylpentanoic Octadecane acid Hexanoic acid Phenethyl alcohol ,6-Di-^r-butyl-4- ^ methylphenol 37 2-Phenethyl butanoate /7-Cresol These compounds were tentatively identified by GC/MS. algae, because the compoundis well knownas a main componentfor the scent of roses. The questions, how phenethyl alcohol is prepared in algae why it increases with decomposition, remain unanswered. But phenethyl alcohol did not seem to play an important role in the rotten odor ofalgae. It is very important that butanol is very abundant in rotten algae. 2-Methylpropanol 3-methylbutanol did not increase as muchas butanol or hexanol, when blue-green algae decomposed. Carboxylic acids also have a strong malodor. Their amounts in algae increased remarkably with decomposition. Acetic propionic acids have an irritating odor but do not play an important role in the malodor. It is likely that 2-methylpropanoic acid is a normal component independent of the decomposition of algae. It is significant that the odor of rotten algae is almost the same as that of butanoic acid, which is a predominant component in volatiles separated from rotten algae. It is reasonable that butanoic acid may be derived microbially from butanol, though obvious evidence of this change has not been obtained yet. It was unexpected that manykinds of esters are present in rotten algae. Also, it was very interesting that esters were not detected in fresh blue-green algae. Generally esters of fatty acids have a fruit-like odor, but their odors could not be clearly detected in rotten algae. These esters probably give the total odor of decomposedalgae a heavy sense. These esters are roughly divided into two classes. One is butanoates the other hexanoates. It is strange that pentyl butanoate or pentyl hexanoate was detected despite no detection of pentanol, that esters of hexanol were not detected despite the presence of hexanol. Overall content ratios of esters were in proportion to ratios of alcohols as follows: butanol 3-methylbutanol > 2-methylpropanol. It has not been determined whether these esters are formed from alcohols or fatty acids by microbial action. Furthermore, acetoin, 2-furfural, 5-methyl- 2-furfural, js-cyclocitral, safranal, acetophenone a,a-dimethylbenzyl alcohol were sometimes detected by GC/MS in fresh bluegreen algae in several additional experiments, but their quantities were small they were not thought to be ordinary components. It was confirmed by these experiments that fresh blue-green algae have few volatile com-

5 Odorous Compoundsin Rotten Blue-green Algae 1765 Table II. Concentrations Odorous Characters of Main Odorous Componentsin Fresh Blue-green Algae (No. 1) Rotten Blue-green Algae (Nos, 2 3) Concentration (/xg/g of dry weight) Compound. Odor quality c oefficient XT No. 1 No.2 xt No. XT 3 Propanol 90% ND 48.7 ND Alcohol-like (P, L) 2-Methylpropanol Alcohol-like (P, L) Butanol 90 ND Fusel oil-like (U, L) l-penten-3-ol 9.3 ND ND Green note (P, L) 3-Methylbutanol Fusel oil-like (U, H) Cyclopentanol 9. 1 ND ND Sweet (P, L) Hexanol 100 ND 84.4 ND Weakly fruity (P, Phenethyl alcohol Rose-like (P, L) L) 2,3-Butanedione ND ND Butter-like (U, L) Fenchone 2.8 ND ND Camphor-like (P, L) Acetic acid Irritative (U, L) Propanoic acid 100 ND Irritative (U, L) 2-Methylpropanoic acid Irritative (U, L) Butanoic acid 98 ND Rancid butter-like (U, H) 3-Methylbutanoic acid 95 ND Stinking sweat-like (U, H) Pentanoic acid 99 ND 2170 ND Stinking sweat-like (U, H) 4-Methylpentanoic acid 98 ND ND 23.0 Stinking sweat-like (U, H) Hexanoic acid 97 ND Haystack-like (U, H) /?-Cresol 90 ND ND Disinfectant-like (U, H) Butyl acetate 72 ND ND 45.4 Pineapple-like (P, L) Ethyl butanoate 74 ND Pineapple-like (P, L) 2-Methylpropyl butanoate 8 1 ND Fruit-like (P, H) Butyl butanoate 87 ND Fruit-like (P, H) 3-Methylbutyl butanoate 90 ND Fruit-like (P, H) Pentyl butanoate 92 ND Apple-like (P, H) Ethyl hexanoate 100 ND Pineapple-like (P, H) 2-Methylpropyl hexanoate 100 ND 14.4 ND Cocoa-like (P, H) Butyl hexanoate 100 ND Pineapple-like (P, H) 3-Methylbutyl hexanoate 98 ND 439 ND Pineapple-like (P, H) 3-Methylbutyl octanoate 100 ND ND 93.9 Fruit-like (P, H) l-penten-3-ol, cyclopentanol fenchone were estimated with recovery coefficients of 90, 92 85%, respectively. ND, not detected; P, pleasant sense; U, unpleasant sense; L, light tone; H, heavy tone. pounds contributing to the malodor. Amounts of malodorous components were larger in sample No. 2 than in sample No. 3. This may suggest that decomposition was not sufficient in sample No. 3 because of the lower temperatures that prohibited any other process of decomposition. Yet both samples had the same organoleptic odor. This observation was explained by the result that content ratios of main components for the malodor did not differ significantly between samples. Therefore, it was concluded from the results of the sensory tests that butanol, butanoic acid, 3-methylbutanoic acid, hexanoic acid butyl butanoate were particularly important contributing factors to the malodor. Esters of butanoic hexanoic acids seemed to give the total odor of rotten algae a heavy sense. A mixed solution of these authentic compounds gave a significantly similar odor to that of rotten blue-green algae. Acknowledgments. Wethank Dr. Takayoshi Kawai for samplings of blue-green algae Dr. Kiyoshi Sugahara for helpful suggestions on the biological aspects of blue-green algae. REFERENCES 1) T. Kikuchi, T. Mimura, K. Harimaya, H. Yano, T. Arimoto, Y. Masada T. Inoue, Chem. Pharm.

6 1766 A. Yasuhara K. Fuwa Bull, 21, 2342 (1973). 2) N. N. Gerber H. A. Lechevalier, Appl. MicrobioL, 13, 935 (1965). 3) T. Kikuchi, T. Mimura, Y. Moriwaki, M. Ando K. Negoro, Chem. Pharm. Bull, 22, 945 (1974). 4) D. Jenkins, L. L. Medsker J. F. Thomas, Environ. Sci. Techno!., 1, 731 (1967).