CARBON-NITROGEN METABOLISM OF SOIL FUNGI V. Role of Cyanide, Nitrite and Sulphur BY S. NATARAJAN* (Department of Chemistry, Madras Christian College, Tambaram) Received February 5, 1958 (Communicated by Dr. S. V. Anantakrislman, r.A.SC.)
INTRODUCTION THE ability of Fusaria to metabolise various carbon and nitrogen sources and grow in media of varying hydrogen-ion concentration has been shown in earlier parts (Natarajan, 1956, 1958). Since both nitrate nitrogen and ammonia nitrogen end up as amino nitrogen in cell proteins, reduction is an essential step. It was expected that by the use of potassium cyanide, potassium nitrite and elementary sulphur, the growth of Fusariurn species would indicate the intermediate stages in the nitrogen utilization. These studies are presented in this communication. MATERIALS AND METHODS The culture of the organisms used in this work was obtained from Centralbureau Voor Schimmelcultures (Baarn). Fusarium vasinfectum and Fusarium udum were grown in Richard's nutrient solution (Rawlins, 1933). The carbon sources were varied by the use of different monosaccharides and disaccharides. With each carbon source, sodium nitrate was used as the nitrogen source. Equivalent quantities of carbon and nitrogen sources were used at the optimum C : N ratio reported in the earlier parts (Natarajan, 1956). The cultural methods employed were similar to those previously described (Natarajan, 1958). Nitrite was detected by the colour reaction of Griess as modified by Blom (1926). Hydrogen sulphide was detected with lead acetate paper strips. Ammonia was detected by Nessler's reagent. The two Fusaria were grown separately under the specified conditions. The culture filtrate was analysed for nitrite and ammonia in each case. The fungi were grown with and without the addition of potassium nitrite, the total nitrogen concentration being kept at 70 mg./50 ml. to study the effect of potassium nitrite; the mat weight and nitrogen in the mat were determined
* Research Assistant, Department of Biochemistry, Indian Institut~ of Science, Bangalore-3, 28 Carbon-Nitrogen Metabolism of Soil Fungi--V 29
TABLE I Toxicity of nitrite on Fusaria in presence of various carbon sources on 20th day of incubation The sum of nitrite nitrogen and nitrate nitrogen is 70 rag. per 50 ml. medium and the amount of nitrite nitrogen is indicated in the table and the balance is nitrate nitrogen.
Nitrite nitrogen/ F. vasinfectum F. udum Carbon 50 ml. source medium Mat weight ~ nitrogen Mat weight ~.nitrogen (mg.) (mg.) in mat (nag.) m mat
Glucose 0 350.9 5.096 314.7 4.700 1 339.2 4.851 300.0 4.512 2 330.0 4.561 294.0 4.316
Fructose 0 600.6 6.135 420.9 5.406 1 590.2 5.946 411.2 5.200 2 580.6 5-528 400.0 5.000
Mannose 0 335.2 4-833 306.2 4-601 1 320.0 4.612 300.0 4.315 2 314.2 4.412 280.1 4.160
Galactose 0 264.0 4.000 180.2 3.546 1 250.0 3.861 171.2 3.220 2 241.9 3.521 166.2 3.000
Sucrose 0 565.4 5.499 360.8 5.211 1 550.2 5.212 340.0 5.000 2 540.3 5.015 330.2 4.815
Maltose 0 370.9 5.299 320.6 4.842 1 360.1 5.012 310.0 4.612 2 345.2 4.812 300.2 4.426
Lactose 0 280.2 4.554 190.5 4.399 1 265.0 4-315 180.2 4.185 2 250.0 4.156 171.5 3.912
on the 20th day of incubation. This was repeated using potassium cyanide in place of nitrite at a concentration of 4 mg./50 ml. and mat weight determined on the 9th day of incubation. The fungi were also grown with the addition of different concentrations (1 and 2 mg./50 ml.) of elementary sulphur, a control also being studied at the same time. The mat weight was noted on the 8th and 16th day of incubation in this series. 30 S. NATARAJAN
DATA AND DISCUSSION When sodium nitrate was used as the nitrogen source with the carbon sources, fructose, sucrose, maltose, mannose, glucose, galactose, lactose, nitrites could be detected in culture filtrate in small quantities, but the greater part was found to be ammonia nitrogen. When 20-day old mats developed on these media were incubated with IX alkaline sodium nitrate, again nitrites were detected. This shows F. vasinfectum and F. udum reduced nitrates to nitrites. Two consecutive reactions may be visualised in the system. ks Nitrite • Ammonia (1) kl Nitrates ~ Nitrites (2) The small concentrations of nitrite coupled with the toxicity of nitrites in larger amounts (Table I) suggest that the reaction (1) is faster than (2). This indicates the presence of a nitrate reductase in the two Fusaria studied. Nitrates may also function as hydrogen acceptors in the presence of the dehydrogenases in Fusaria (Nord and Mull, 1945). In the presence of potassium cyanide our observations showed that the growth of F. vasinfectum TABLE II The influence of added potassium cyanide on Fusaria in presence of various carbon sources on the 8th day of incubation 4 rag. KCN is added after 4 days growth
F. vasinfectum F. udum Carbon source Mat weight Mat weight Mat weight Mat weight (mg.) (rag.) (rag.) (rag.) without KCN with KCN without KCN with KCN
Glucose 260.2 261.0 180.7 181.1 Fructose 320.2 319.2 240.9 241.9 Mannose 250"2 248.9 165.8 164-7 Galactose 135.2 136.1 94.3 95.0 Sucrose 284" 6 285-1 218.6 219.0 Maltose 275"2 274.8 190.4 190.0 Lactose 140.4 141.1 100.2 100.5 Carbon-Nitrogen Metabolism of Soil Fungi--V 31 and F. udum was unaffected by the potassium cyanide in the concentrations used (Table II). Apparently a potassium cyanide insensitive 'Nitrate re- ductase' is present. Nitrite was detected under these conditions too. When the fungi were grown in presence of nitrite a marked decrease in mat weight and percent nitrogen in mat were noticed (Table I). The addition of elementary sulphur led to positive tests for hydrogen sulphide during growth of both Fusaria indicating that sulphur acts as a hydrogen acceptor in the medium used and no nitrite could be detected in the culture. The growth is better, as shown by mat weight (Table III). It is therefore possible
TABLE Ill The influence of added sulphur on Fusaria in presence of various carbon sources
Mat weight (mg.) Sulphur Carbon added/50 ml. F. vasinfectum F. udum source medium (mg.) Incubation Incubation 8 days 16 days 8 days 16 days
Fructose 0 320.2 560.8 240.9 390.8 1 326.8 570.1 248.2 400.1 2 330.1 580.1 260-1 408-2 Sucrose 0 284.6 525.2 218.6 320.4 1 290.1 535.1 226.1 330.2 2 300.1 548.1 230.2 340.1 Maltose 0 275.2 355.2 190-4 295.4 1 280.7 360-7 200.2 305.8 2 285 "2 364.5 208.3 309.1 Glucose 0 260.9 331.4 188.7 280.8 1 265-1 340.2 190.2 290.3 2 270.2 348.2 196-7 296.4 Mannose 0 250.2 320.5 165.8 275.4 1 260.2 328.2 172.4 286.8 2 265.6 334.2 181.4 290.4 Lactose 0 140.4 248.4 100.2 165.2 1 146.8 259.1 108.6 170.3 2 150.2 264-3 112.3 180.2 Galactose .. 0 135.3 240.1 94.3 161.3 1 140-1 251-2 98-4 166.6 2 146.2 260.1 108.2 172-4 32 S. NATARAJAN
that the apparent absence of nitrites in the presence of sulphur is due to its functioning as an oxidizing agent towards hydrogen sulphide. In other words the reaction between elementary sulphur and dehydrogenases in Fusaria can be represented by the following equations: S ° + 2H = H~S 2H~S + 02 = 2S + 2H~O 2H20 + H2S + 40 = SO4=+ 2HzO +.
SUMMARY Fusaria reduce nitrates to nitrite and ammonia irrespective of the carbon source. Fusaria possess potassium cyanide insensitive nitrate reductase.
ACKNOWLEDGEMENTS I wish to express my sincere thanks to Professors Dr. S. V. Ananta- krishnan and Dr. T. S. Sadasivan for their suggestions and guidance.
LITERATURE CITED
1. Blom, J. .. "Sensitive and Specific Reaction for Nitrate and for Hydoxylamine," Ber., 1926, 59, 121-25. 2. Natarajan, S. .. "Carbon-Nitrogen Metabolism of Soil Fungi--I," Proc. Indian Acad. Sci., 1956, 44B(5), 289-99. 3. .. "Carbon-Nitrogen Metabolism of Soil FungimlI," 1bid., 1956, 44 B (5), 300-10. 4. .. "Carbon-Nitrogen Metabolism of Soil Fungi--IlI," J. Madras Univ., 1956, 2~iB(3), 629-38. 5. .. "Carbon-Nitrogen Metabolism of Soil Fungi--IV. Influ- ence of Carbon Sources," Proc. lndian Acad. Sci., 1958, 47 B, 338. 6. Nord, F. F. and Mull, "Recent Progress in the Biochemistry of Fusaria," Adrances R.P. in Enzymology, 1945, 5, 165-205. 7. Raw/ins, T.E. .. Phytopathologieal and Botanical Research Methods, N.Y., John Wiley & Sons Ltd., London, 1933.