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Influence of temperature on lake bacterial activity
Water Research Vol. 8. pp. 525 to 528. Pergamon Preul, 197~. Printed in Great Britain.
I N F L U E N C E OF T E M P E R A T U R E ON LAKE BACTERIAL ACTIVITY S. S. Rno and B. J. Dua'Kn Microbiology Laboratories, Canada Centre for Inland Waters, Burlington, Ontario, Canada (Received 12 January 1974)
Abstract---Oxygen utilization rates of isolates of Flavobacterium from Lake Ontario and Lake Superior, and an E. coli from the St. Lawrence River were observed at 4 and 20"C. Data presented indicate that the oxygen utilization rate of the lake bacteria at 4°C is similar to that of the river bacteria at 20°C. The observation is also extended to explain the seemingly satisfactory biodegradation of nutrients discharged into water bodies in temperate climates. INTRODUCTION effect of low temperatures on the behaviour of aquatic Studies on microbial biodegradation of nutrient input bacteria. Hobbie et al. (1972) and Morita (1973) have into lakes and rivers have been receiving greater atten- indicated that depth and low temperature influence tion and recognition because of the accelerated eutro- microbial activity however, there is a paucity of data phication or aging of some of our waters (Kuznetsov, available in this area. 1968; Lee and Fruh, 1966). The biodegradation process During the course of some studies on the distribution in this instance refers to the conversion of more of microbial species in Lake Superior an interesting complex organic materials into one or more simpler observation of bacterial activity at low temperature was substances by microbial agents. The trophic state noted. The results are briefly presented here. of any lake appears to be related to nutrient loading i.e. nitrogen and phosphorus from sewage, industrial wastes EXPERIMENTAL and land runoff (Srinath and Pillai, 1966; Oswald and Water samples collected in May, 1-m below the Golueke, 1966) and also in temperate climates to the depth and volume of the lake (Vollenweider, 1968). In surface, from the western end of Lake Ontario, near addition, the importance of micro-organisms in the bio- Burlington, and from the north shore of Lake Superior, degradation of these inputs and the changing of the near Marathon, were used in this investigation. Immebiochemical complexity and trophicity of receiving diately upon returning to the laboratory (Burlington, waters has been shown by Alexander (1971) and Soro- Ontario) a portion of the water sample, which was to be used as the substrate in respiration studies, was autokin (1971). The basic principles of bacterial growth and meta- claved (121°C for 20 min) and then analyzed for some bolism in a nutritionally adequate environment at basic nutrients (Table 1). Another portion was plated optimum or near optimum temperatures are well for isolation of the numerically dominating heteroestablished (Hendricks, 1972). However, growth, multi- trophic bacterial species. Bacterial growth medium used plication a n d maintenance of bacteria in the aquatic was Peptone, 0-5 g; yeast extract, 0-1 rag; soluble casein, environment, where temperature and available nutri- 0.5 g; K2HPO,t, 0.2 g; MgSO4"7H20, 0.05 g; FeC13, ents are far from optimum, has not yet been clearly defined (Hendricks, 1972). Aquatic bacteria are also Table 1. Basic nutrient analysis of autoclaved Lake Ontario water (western end) subjected to multiple external influences such as the fluctuating input of interfering substances in industrial Item Concentration waste discharges, sunlight and turbidity. Initial studies in aquatic microbiology were quantiTotal phosphorous (P) 152/~g 1-t tative enumerations of seasonal distributions of bacteria Soluble reactive phosphorous (P) 92/zg 1- t in small lakes (Collins, 1963) and their relationship to Soluble organic carbon (C) 3700/~g 1- t 45/~g 1- t the nutrient status of the lake system (Kuznetsov, 1968). Ammonia nitrogen (N) Nitrite and nitrate nitrogen (N) 410/Jg 1-t Although these and similar observations have added Sulphate 31 ~g l- t considerably to the general understanding of bacterial pH 7-2 ecology, further studies are required to evaluate the 525-.
526
S.S. RAo and B. J. DUTKA
0.005 g; Bacto agar, 15 g: and distilled water, I 1. The final pH of the medium was 7-2. Incubation was at 20°C for 7 days. The numerically dominant species, from both water samples, were identified as Flavobacterium. The characterstics of these are given in Table 2. Table 2. Biochemical characteristics of the flavobacterium isolated from Lake Ontario and Lake Superior Non-diffusible pigment Gram reaction Appearance Motility Catalase Oxidase Glucose OF Lactose Citrate MacConkey MR V-P
Yellow Negative Rod + + 0
+
Indole Nitrate reduction Gelatin Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase
RESULTS AND DISCUSSION
The importance of various bacterial species in the biocoenosis within the ecosystem of a body of water is now being realised. In considering bacterial species as important agents in the biodegradation of organic material, their behaviour under varied environmental conditions must be clearly defined. In temperate climates for instance, temperature fluctuations may greatly influence bacterial activity. However, such wide temperature fluctuations are seldom seen in the Great Lakes since the volume of the lakes precludes a wide seasonal temperature variation. Data presented in Figs. I-3 indicate that a relationship exists between temperature and oxygen uptake rates. The Flavobacterium isolates had greater oxygen uptake rates at 4 than at 20°C and thus could be considered as psycrotolerant or psycrophilic (Gordon, 1971). In contrast, the E. coil isolate produced the expected typical pattern of a mesophile, of greater oxygen uptake at 20°C. Hendricks (1971) also observed results similar to those of E. coli during respiration studies with enteric bacterial isolates, using as substrate autoclaved river
0t
Respiration studies, to observe oxygen uptake rates at 4 and 20°C, were performed in duplicate using a Gilson Differential Respirometer, nutrient free washed bacterial cells (107 ml - t ) and autoclaved lake water as substrate. These temperatures were chosen as the majority of the Great Lakes waters are usually at or near 4°C and seldom reach 20°C (Blaise, 1973). Organism respiration has been used in this study as an index of in situ activity. Bacterial cells grown overnight on the basal plating medium were harvested and washed three times in sterile distilled water by refrigerated centrifugation at 10,000rev min- ' for 20rain ina IEC B-20 model centrifuge. Each Warburg flask contained 0-5 ml of a standardized rested bacterial suspension having a turbidity value of 18 (this value was obtained by using a Klett Summerson Photoelectric Coiorimeter with a blue filter at 420/an), 1 ml of sterilized lake water (substrate) and 0-2 ml of 20 per cent KOH in the centre well for CO2 absorption. A final volume of 3 ml was maintained in the flask by adding distilled water. A control flask containing 1 ml of sterilized lake water, 2 ml distilled water and 0.2 ml of 20 per cent KOH in the centre well was also used. A pre-incubation period of 20 rain was used in order to bring the temperature of the flask contents to that of the waterbath. A similar study was performed using an E. coil isolate from the St. Lawrence River.
• 4°
~601
o20"
0
Fig. l.
30
60
90 TIME, rain
120
150
180
Effect of temperature on the oxygen utilization by a
Flovobacterium isolated from Lake Ontario.
0
30
60
90 TIME, rain
120
150
180
Fig. 2. Effectof temperature on the oxygenutilization by a Flaoobacterium isolated from Lake Superior.
527
Influence of temperature on lake bacterial activity
8, I
:
7"
~
~jZ20
Q
•
0
30
60
90 TIME,
120
150
~80
of our cold oligotrophic lakes varies little from season to season. Studies are currently underway to try and relate the effect of some physical factor, such as temperature, pH, dissolved oxygen on the physiological bebaviour of several bacterial isolates, collected during different seasons from various lakes, in lake water, sediment eluates and some industrial effluents. These studies should provide an insight into some of the principles relating to bacteriological dynamics in relation to cold water lake eutrophication.
rnin
Fig. 3. Effectof temperature on the oxygen utilizationby an E. coli isolatedfrom St. Lawrence River. water and sediment eluates. In Hendricks' study area, the river water temp. varied between 18-20°C for the major part of the year, thus acclimatization of the river organism to these temperatures is expected. However, lake bacteria, because of their acclimation or adaptation to colder temperatures, 3-10°C, for long periods of time perhaps behave differently. Figures 1-3 data also indicate that the rate of oxygen utlization by Flavobacterium at 4°C is similar to that of E. coli at 20°C. The presence of psycrophilic or psycrotoleram populations in bodies of water which have temperatures below the optimum for mesophilic type bacterial respiration may account for the seemingly satisfactory rate of biodegradation of waste organic matter which is discharged into them. These organisms may also have a selective advantage over facultative psycrophiles in the relatively constant low temperature environment of the Great Lakes. As the assimilative capacity of a body of water depends on microbial degradation of nutrient input, which is apparently related to temperature, the Flavobacterium data suggest that through their higher activity at lower temperatures, nutrient utilization is perhaps more effective at lower temperature Flavobacterium were found in large numbers in Lake Ontario and their proportion percentage increased for 1971-1972 (Seyfried, 1973). These observations support the hypotheses that the bulk of biodegradation of pollutants in aquatic systems mast be performed by organisms able to grow and metabolize at temperatures below 5°C (Blaise, 1973). Also the present data could be reasonably extended to explain biodegradation of waste input in some of our Great Lakes and probably some of the rivers where temperature and nutrient concentration are well below optimum, for mesophiles. Lake Superior, for example even at its warmest, seldom has inshore surface temp. reaching 12-15°C. Perhaps the assimilative capacity
Acknowledgements--Authors wish to thank the water quality subdivision personnel, CCIW, for their assistance in the chemical analysis of the water sample, Dr M. Munawar, Great Lakes BiolimnologYSubdivision, Dr N. M. Bums, Descriptive Limnology Subdivision, and Dr C. W. Hendricks, Department of Microbiology, Universityof Georgia, Athens, Georgia, for their useful criticism.
REFERENCES
Alexander M. (1971) Microbial Ecology. p. 5l 1. Wiley, New York. Blaise C. R. (1973) Degradative microorganisms in the Ottawa River. A Thesis submitted in partial fulfillmentof the requirements for the degree of Master of Science at the University of Ottawa, p. 24. Collins V, G. (1963)The distribution and ecologyof bacteria in fresh water. Proc. Soc. Water Treat. Exam. 12, 40-73. Lee G. F. and Fruh E. G. (1966) Reprinted from the U.S. Dept. of Interior, Federal Water Pollution Control Administration, Washington, D.C. 20203, from Industrial Water Engineering, Feb. 1966. Gordon R. C. (197l) Depletion of oxygenby microorganisms in Alaskan rivers at low temperatures. Int. Syrup. War. Pollut. Control in Cold Climates, Univ. of Alaska, July 22-24, 1970pp. 71-95. Hendricks C. W. (1971) Enteric bacterial metabolism of stream sediment eluates. Can. J. Microbiol., 17, 551-556. Hendricks C. W. (1972) Microbial Degradation of Oil Pofiutants. A workshop sponsored by the Office of Naval Research. The U.S. Coast Guard, The EPA and Georgia State University. Dec. 4-6, 1972, p. 57. Hobble E. H., Hansen O. H., Packard T. T., Pomeroy L. R., Sheldon R. W., Thomas J. P. and Wieb¢ W. J. (1972) A study of the distribution and activity of microorganisms in ocean water. Limnol. Oceanogr. 17, 544-555. lngraham J. L. (1973) Growth of Bacteria in Low Temperature Environments. 1st Int. Cong. Bact. Jerusalem Sept. 27 Alas. Vol. 1, 121. Kuznetsov S. I. (1968) Recent studies on the role of microorganisms in the cycling of substances in lakes. LimnoL Oceanogr., 13, 211-224. Morita R. Y. (1973) Hydrostatic pressure, temperature and salanity effects on marine micro-organisms. 1st Int. Cong. Bact. Jerusalem, Sept. 2-7 Abs. Vol. 1, 114. Oswald W. J. and Golueke C. G. (1966) Eutrophication trends in the United States--a problem. J. Wat. Pollut. Control Fed., 38, 964-975. Seyfried P. L. (unpublished data) Dept. Microbiology, School of Hygiene University of Toronto.
528
S.S. RAo and B. J. DU'rg.A
Sorokin Yu. I. (1971) Bacterial populations as components of oceanic ecosystem. Marine Biol., 11, 10[-[05. Srinath E. G. and Pillal S. C. (1966) Phosphorous in sewage, polluted waters, sludges and e~luents. Quart. Rev. Biol., 41, 384-407.
Vollenweider R. A. (1968) Scientific fundamentals of the eutrophication of lakes and flowing waters with particular reference to nitrogen and phophorous as factors in eutro. ph/cat/on. Organization for Economic Cooperation and Development Directorate for Scientific Affairs.
I N F L U E N C E OF T E M P E R A T U R E ON LAKE BACTERIAL ACTIVITY S. S. Rno and B. J. Dua'Kn Microbiology Laboratories, Canada Centre for Inland Waters, Burlington, Ontario, Canada (Received 12 January 1974)
Abstract---Oxygen utilization rates of isolates of Flavobacterium from Lake Ontario and Lake Superior, and an E. coli from the St. Lawrence River were observed at 4 and 20"C. Data presented indicate that the oxygen utilization rate of the lake bacteria at 4°C is similar to that of the river bacteria at 20°C. The observation is also extended to explain the seemingly satisfactory biodegradation of nutrients discharged into water bodies in temperate climates. INTRODUCTION effect of low temperatures on the behaviour of aquatic Studies on microbial biodegradation of nutrient input bacteria. Hobbie et al. (1972) and Morita (1973) have into lakes and rivers have been receiving greater atten- indicated that depth and low temperature influence tion and recognition because of the accelerated eutro- microbial activity however, there is a paucity of data phication or aging of some of our waters (Kuznetsov, available in this area. 1968; Lee and Fruh, 1966). The biodegradation process During the course of some studies on the distribution in this instance refers to the conversion of more of microbial species in Lake Superior an interesting complex organic materials into one or more simpler observation of bacterial activity at low temperature was substances by microbial agents. The trophic state noted. The results are briefly presented here. of any lake appears to be related to nutrient loading i.e. nitrogen and phosphorus from sewage, industrial wastes EXPERIMENTAL and land runoff (Srinath and Pillai, 1966; Oswald and Water samples collected in May, 1-m below the Golueke, 1966) and also in temperate climates to the depth and volume of the lake (Vollenweider, 1968). In surface, from the western end of Lake Ontario, near addition, the importance of micro-organisms in the bio- Burlington, and from the north shore of Lake Superior, degradation of these inputs and the changing of the near Marathon, were used in this investigation. Immebiochemical complexity and trophicity of receiving diately upon returning to the laboratory (Burlington, waters has been shown by Alexander (1971) and Soro- Ontario) a portion of the water sample, which was to be used as the substrate in respiration studies, was autokin (1971). The basic principles of bacterial growth and meta- claved (121°C for 20 min) and then analyzed for some bolism in a nutritionally adequate environment at basic nutrients (Table 1). Another portion was plated optimum or near optimum temperatures are well for isolation of the numerically dominating heteroestablished (Hendricks, 1972). However, growth, multi- trophic bacterial species. Bacterial growth medium used plication a n d maintenance of bacteria in the aquatic was Peptone, 0-5 g; yeast extract, 0-1 rag; soluble casein, environment, where temperature and available nutri- 0.5 g; K2HPO,t, 0.2 g; MgSO4"7H20, 0.05 g; FeC13, ents are far from optimum, has not yet been clearly defined (Hendricks, 1972). Aquatic bacteria are also Table 1. Basic nutrient analysis of autoclaved Lake Ontario water (western end) subjected to multiple external influences such as the fluctuating input of interfering substances in industrial Item Concentration waste discharges, sunlight and turbidity. Initial studies in aquatic microbiology were quantiTotal phosphorous (P) 152/~g 1-t tative enumerations of seasonal distributions of bacteria Soluble reactive phosphorous (P) 92/zg 1- t in small lakes (Collins, 1963) and their relationship to Soluble organic carbon (C) 3700/~g 1- t 45/~g 1- t the nutrient status of the lake system (Kuznetsov, 1968). Ammonia nitrogen (N) Nitrite and nitrate nitrogen (N) 410/Jg 1-t Although these and similar observations have added Sulphate 31 ~g l- t considerably to the general understanding of bacterial pH 7-2 ecology, further studies are required to evaluate the 525-.
526
S.S. RAo and B. J. DUTKA
0.005 g; Bacto agar, 15 g: and distilled water, I 1. The final pH of the medium was 7-2. Incubation was at 20°C for 7 days. The numerically dominant species, from both water samples, were identified as Flavobacterium. The characterstics of these are given in Table 2. Table 2. Biochemical characteristics of the flavobacterium isolated from Lake Ontario and Lake Superior Non-diffusible pigment Gram reaction Appearance Motility Catalase Oxidase Glucose OF Lactose Citrate MacConkey MR V-P
Yellow Negative Rod + + 0
+
Indole Nitrate reduction Gelatin Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase
RESULTS AND DISCUSSION
The importance of various bacterial species in the biocoenosis within the ecosystem of a body of water is now being realised. In considering bacterial species as important agents in the biodegradation of organic material, their behaviour under varied environmental conditions must be clearly defined. In temperate climates for instance, temperature fluctuations may greatly influence bacterial activity. However, such wide temperature fluctuations are seldom seen in the Great Lakes since the volume of the lakes precludes a wide seasonal temperature variation. Data presented in Figs. I-3 indicate that a relationship exists between temperature and oxygen uptake rates. The Flavobacterium isolates had greater oxygen uptake rates at 4 than at 20°C and thus could be considered as psycrotolerant or psycrophilic (Gordon, 1971). In contrast, the E. coil isolate produced the expected typical pattern of a mesophile, of greater oxygen uptake at 20°C. Hendricks (1971) also observed results similar to those of E. coli during respiration studies with enteric bacterial isolates, using as substrate autoclaved river
0t
Respiration studies, to observe oxygen uptake rates at 4 and 20°C, were performed in duplicate using a Gilson Differential Respirometer, nutrient free washed bacterial cells (107 ml - t ) and autoclaved lake water as substrate. These temperatures were chosen as the majority of the Great Lakes waters are usually at or near 4°C and seldom reach 20°C (Blaise, 1973). Organism respiration has been used in this study as an index of in situ activity. Bacterial cells grown overnight on the basal plating medium were harvested and washed three times in sterile distilled water by refrigerated centrifugation at 10,000rev min- ' for 20rain ina IEC B-20 model centrifuge. Each Warburg flask contained 0-5 ml of a standardized rested bacterial suspension having a turbidity value of 18 (this value was obtained by using a Klett Summerson Photoelectric Coiorimeter with a blue filter at 420/an), 1 ml of sterilized lake water (substrate) and 0-2 ml of 20 per cent KOH in the centre well for CO2 absorption. A final volume of 3 ml was maintained in the flask by adding distilled water. A control flask containing 1 ml of sterilized lake water, 2 ml distilled water and 0.2 ml of 20 per cent KOH in the centre well was also used. A pre-incubation period of 20 rain was used in order to bring the temperature of the flask contents to that of the waterbath. A similar study was performed using an E. coil isolate from the St. Lawrence River.
• 4°
~601
o20"
0
Fig. l.
30
60
90 TIME, rain
120
150
180
Effect of temperature on the oxygen utilization by a
Flovobacterium isolated from Lake Ontario.
0
30
60
90 TIME, rain
120
150
180
Fig. 2. Effectof temperature on the oxygenutilization by a Flaoobacterium isolated from Lake Superior.
527
Influence of temperature on lake bacterial activity
8, I
:
7"
~
~jZ20
Q
•
0
30
60
90 TIME,
120
150
~80
of our cold oligotrophic lakes varies little from season to season. Studies are currently underway to try and relate the effect of some physical factor, such as temperature, pH, dissolved oxygen on the physiological bebaviour of several bacterial isolates, collected during different seasons from various lakes, in lake water, sediment eluates and some industrial effluents. These studies should provide an insight into some of the principles relating to bacteriological dynamics in relation to cold water lake eutrophication.
rnin
Fig. 3. Effectof temperature on the oxygen utilizationby an E. coli isolatedfrom St. Lawrence River. water and sediment eluates. In Hendricks' study area, the river water temp. varied between 18-20°C for the major part of the year, thus acclimatization of the river organism to these temperatures is expected. However, lake bacteria, because of their acclimation or adaptation to colder temperatures, 3-10°C, for long periods of time perhaps behave differently. Figures 1-3 data also indicate that the rate of oxygen utlization by Flavobacterium at 4°C is similar to that of E. coli at 20°C. The presence of psycrophilic or psycrotoleram populations in bodies of water which have temperatures below the optimum for mesophilic type bacterial respiration may account for the seemingly satisfactory rate of biodegradation of waste organic matter which is discharged into them. These organisms may also have a selective advantage over facultative psycrophiles in the relatively constant low temperature environment of the Great Lakes. As the assimilative capacity of a body of water depends on microbial degradation of nutrient input, which is apparently related to temperature, the Flavobacterium data suggest that through their higher activity at lower temperatures, nutrient utilization is perhaps more effective at lower temperature Flavobacterium were found in large numbers in Lake Ontario and their proportion percentage increased for 1971-1972 (Seyfried, 1973). These observations support the hypotheses that the bulk of biodegradation of pollutants in aquatic systems mast be performed by organisms able to grow and metabolize at temperatures below 5°C (Blaise, 1973). Also the present data could be reasonably extended to explain biodegradation of waste input in some of our Great Lakes and probably some of the rivers where temperature and nutrient concentration are well below optimum, for mesophiles. Lake Superior, for example even at its warmest, seldom has inshore surface temp. reaching 12-15°C. Perhaps the assimilative capacity
Acknowledgements--Authors wish to thank the water quality subdivision personnel, CCIW, for their assistance in the chemical analysis of the water sample, Dr M. Munawar, Great Lakes BiolimnologYSubdivision, Dr N. M. Bums, Descriptive Limnology Subdivision, and Dr C. W. Hendricks, Department of Microbiology, Universityof Georgia, Athens, Georgia, for their useful criticism.
REFERENCES
Alexander M. (1971) Microbial Ecology. p. 5l 1. Wiley, New York. Blaise C. R. (1973) Degradative microorganisms in the Ottawa River. A Thesis submitted in partial fulfillmentof the requirements for the degree of Master of Science at the University of Ottawa, p. 24. Collins V, G. (1963)The distribution and ecologyof bacteria in fresh water. Proc. Soc. Water Treat. Exam. 12, 40-73. Lee G. F. and Fruh E. G. (1966) Reprinted from the U.S. Dept. of Interior, Federal Water Pollution Control Administration, Washington, D.C. 20203, from Industrial Water Engineering, Feb. 1966. Gordon R. C. (197l) Depletion of oxygenby microorganisms in Alaskan rivers at low temperatures. Int. Syrup. War. Pollut. Control in Cold Climates, Univ. of Alaska, July 22-24, 1970pp. 71-95. Hendricks C. W. (1971) Enteric bacterial metabolism of stream sediment eluates. Can. J. Microbiol., 17, 551-556. Hendricks C. W. (1972) Microbial Degradation of Oil Pofiutants. A workshop sponsored by the Office of Naval Research. The U.S. Coast Guard, The EPA and Georgia State University. Dec. 4-6, 1972, p. 57. Hobble E. H., Hansen O. H., Packard T. T., Pomeroy L. R., Sheldon R. W., Thomas J. P. and Wieb¢ W. J. (1972) A study of the distribution and activity of microorganisms in ocean water. Limnol. Oceanogr. 17, 544-555. lngraham J. L. (1973) Growth of Bacteria in Low Temperature Environments. 1st Int. Cong. Bact. Jerusalem Sept. 27 Alas. Vol. 1, 121. Kuznetsov S. I. (1968) Recent studies on the role of microorganisms in the cycling of substances in lakes. LimnoL Oceanogr., 13, 211-224. Morita R. Y. (1973) Hydrostatic pressure, temperature and salanity effects on marine micro-organisms. 1st Int. Cong. Bact. Jerusalem, Sept. 2-7 Abs. Vol. 1, 114. Oswald W. J. and Golueke C. G. (1966) Eutrophication trends in the United States--a problem. J. Wat. Pollut. Control Fed., 38, 964-975. Seyfried P. L. (unpublished data) Dept. Microbiology, School of Hygiene University of Toronto.
528
S.S. RAo and B. J. DU'rg.A
Sorokin Yu. I. (1971) Bacterial populations as components of oceanic ecosystem. Marine Biol., 11, 10[-[05. Srinath E. G. and Pillal S. C. (1966) Phosphorous in sewage, polluted waters, sludges and e~luents. Quart. Rev. Biol., 41, 384-407.
Vollenweider R. A. (1968) Scientific fundamentals of the eutrophication of lakes and flowing waters with particular reference to nitrogen and phophorous as factors in eutro. ph/cat/on. Organization for Economic Cooperation and Development Directorate for Scientific Affairs.