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Biodegradation of paddy straw with cellulolytic fungi and its application on wheat crop
Bioresource Technology 47 (1994) 185-188
Short Communication
Biodegradation of Paddy Straw with Cellulolytic Fungi and its Application on Wheat Crop
METHODS The C : N ratio of chopped, air-dried paddy straw was adjusted to 60:1 by adding nitrogen as urea. Rock phosphate (1% w/w) was also added. The moisture content was adjusted to 80%. High-density polythene bags were filled with 2 kg of the material. Small holes were made throughout the bags for aeration. Before the residue was put into the bags it was inoculated with the selected four fungi singly or in combination according to the following treatment schedule:
Abstract Chopped paddy straw was used as a substrate to study the pattern of biodegradation after inoculation (singly or in combination) with four cellulolytic fungi: Trichoderma reesei QM 9414, T. viride, Pleurotus sajor-caju and Coprinus cinereus. To assess the rate of biodegradation, samples were drawn at regular intervals and analysed for organic carbon, total nitrogen, humus synthesis and respiratory activities of the degrading substrate. The C:N ratio was also calculated to judge the extent of biodegradation. Maximum lowering of the C: N ratio was observed in the treatment where dual inoculation with T. reesei and P. sajor-caju was done. A pot-culture experiment was conducted to assess the effects of application of composted paddy straw on the yield of wheat. Application of the paddy straw degraded by' the two fungi, in combination with urea at 80 kg N ha i, produced a significant effect on the yield of wheat.
( 1) (2) (3) (4) (5)
Trichoderma reesei QM 9414 Trichoderma viride 1433 Pleurotus sajor-caju Coprinus cinereus syn lagopus 3545 Trichoderma reesei QM 9414 + Pleurotus sajorcaju (6) Trichoderma reesei QM 9414 + Coprinus cinereus
(7) Trichoderma viride 1433 + Pleurotus sajor-caju (8) Trichoderma viride 1433 + Coprinus cinereus (9) Control -- uninocutated Each treatment was replicated three times. The inoculum was prepared according to the method of Kahlon and Dass (1987). The straw was inoculated with the respective inocula at 0"1% w/w (2 g/2 kg substrate). In treatments where two cultures were added, the total quantity of the inoculum (i.e. 0-1%) was kept constant and the two organisms were added in equal proportions. The temperature varied between 31"7°C and 24"8°C during the degradation period. The inoculated bags were kept in a glasshouse and sampling was done at intervals of 20, 40 and 60 days. Chemical and microbiological analyses of these samples were made to monitor the changes.
Key words': Paddy straw, cellulolytic fungi, biodegradation. INTRODUCTION The total production of rice in India is 7"5 × 107 t, which is 9"3% of the world production. Thus, approximately double this amount of paddy straw is generated every year (Economic Survey of India, 1990-1991) and disposal poses several problems. Much straw is burnt, but this has been criticised because of air pollution and respiratory problems and the danger of soil erosion due to repeated burning (Kahlon & Dass, 1987). Paddy straw could be used as an animal feed; however, it has not been popular. The major limitations as feed are low digestibility, low protein content and high lignin and silica contents. An alternative use for paddy straw could be its biodegradation to organic manure. In the present study, paddy straw was biodegraded with four cellulolytic fungi (singly or in combination) and this composted straw was applied as an organic manure to assess its effect on the yield of wheat.
Chemical and microbiological analyses The samples were dried at 60°C and ground to pass a 30 mesh (1 mm) sieve. A sample was ashed at 600°C for 4 h and the total carbon was taken to be organic matter (%)x 0"56 (Anon., 1953). Total nitrogen was determined by a Kjeldahl method (Jackson, 1967) using an autoanalyser. Humus content was estimated by the method of Kononova (1966). The rate of decomposition of paddy straw was determined by estimating the amount of carbon dioxide evolved during the process by the method of Pramer and Schmidt (1964).
Bioresource Technology 0960-8524/94/S07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain 185
186
Sarojni Kanotra, R. S. M a t h u r
Effect of biodegraded material on plant growth and yield A pot culture experiment was conducted to test the effect of application of biodegraded material prepared by cellulolytic action of selected fungi on the yield of a wheat crop (vat. HD 2285). The soil used was loamy in texture, alkaline (pH 8-5), low in organic carbon (0"38%) and total nitrogen (0"028%) contents. The quantity of available phosphorus and potash was 13"5 and 179-0 kg/h respectively. The pot-culture experiment was conducted during the winter season of 1988-1989. The temperature ranged from 6°C to 25°C during the season. The composted material prepared by the inoculation of efficient cellulolytic fungi was applied at 10 t/ha. It was mixed thoroughly with 10 kg of air-dried sieved soil. Inorganic nitrogenous fertilizer as urea was applied at 80 kg of N/ha in all the treatments except one where 120 kg N/ha was applied. A basal dressing of phosphatic
fertilizer (as single superphosphate at 60 kg P2Os/ha ) and potassic fertilizer (as muriate of potash at 40 kg P2Os/ha) was also given. A control was kept in which no fertilizer was added. The treatment schedule is seen in Fig. 1. Each treatment was replicated three times. After harvesting, plants were dried in an oven at 65°C for 72 h, and the yield of grain and straw was recorded. Statistical methods The data were analysed statistically wherever needed by using a random block design as outlined in Panse and Sukhatme (1978) and the test of significance was done at the 5% level. RESULTS AND DISCUSSION During biodegradation of paddy straw the organic carbon decreased gradually, and 10% of the control straw was lost in 60 days, while the loss was greater
55--
Nso+PADDY STRAW BIODEGRADEDBY []
50--
[] [] []
25
o ct.
[] [] []
m
v a .._I
I
15
).-
UNINOCULATED TRICHODERMA REESEI TRICHODERMA VIRIDE PLEUROTUS SAJOR-CAJU COPRINUS CINEREUS 1".REESEI -I-R SAJOR- CAJU I".REESEI-t-C.CINEREUS T.VIRIDE'F R SAJOR- CAdU T.VIRIDE-I- C, CINEREUS
Z c¢ I0 0 5
0 NO
N80 NI20
Effect of paddy straw biodegraded by cellulolytic fungi on grain yield of wheat (HD 2285).
Fig. l.
Table 1. Changes in C: N during biodegradation of paddy straw by ceilulolytic fungi Paddy straw inoculated with
Uninoculated Trichoderma reesei QM 9414 7". viride Pleurotus sajor-caju Coprinus cinereus T. reesei Q M 9414+ P. sajor-caju T. reesei QM 9414 + C. cinereus T. viride + P. safor-caju T. viride + C. cinereus
C.D. at 5% Amended C : N = 60 : 1.
Sampling time 20 Days
40 Days
60 Days
55"86 29-24 43"97 45"30 45"83 32-79 38"16 33"40 59"00
39'56 22'10 26'04 26'25 26'52 19'79 29"93 20"94 27"63
33"61 19'30 24"20 23"20 25'13 18"05 20'91 21'06 25'35
5"06
2"75
2.00
Biodegradation of paddy straw with fungi PADDY STRAW INOCULATED WITH
35-
[] [Im I~ [] [] [] [] [] []
30-
25-
o~ CO
187
20-
xxxx
UNINOCULATED TRICHODERMAREESEI TRICHODERMAVIRIOE PLEUROTUS SAOOR-CAJU COPRINUS CINEREUS T.REESEI+ P. SAJOR-CAJU T.REESEI+C.CINEREUS T.VIRIDE+ RSAJOR-CAJU T.VIRIDE+ C.CINEREUS
-r" 15 xxxx xxxx
I0 \XNN
5
0
II SINGLE INOCULATION
:ONTROL
Humus content (%) in paddy straw biodegraded by cellulolytic fungi inoculated singly or in combination.
Fig. 2.
PADDY STRAW INOCULATED WITH 2000
H UNINOCULATED 0-..-0 T.REESEI QM 9414
1800
-" "-- p. SAJOR--CAJU ,,'~ &-- -.~ T.REESEI+ -," RSAJOR/~" CAJU /
W
1600
I-
1400
//
/
I11 if)
,,~
/
1200
/
)~'
/
I000 W
0> N
0 o
800 60O 4O0 200 0
0
I0
20
30
40
50
60
70
DAYS OF INOCULATION
Fig. 3.
DUAL INOCULATION
Cumulative CO~ evolved during degradation of paddy straw inocuiated with cellulolytic fungi.
with fungal inoculation; Trichoderma reesei QM 9414 plus Pleurotus sajor-caju showed the maximum decomposition of 33%. The total nitrogen content showed an increase, the increase being maximum in the same treatment. Hence, both the fungal cultures together brought down the C:N ratio of paddy straw to 18:1 within 60 days (Table 1 ). The humus synthesis also was improved by dual inoculation of T. reesei and P. sajorcaju in comparison to the control (Fig. 2). The maximum (32"2%) humus was observed in this treatment, after an interval of 60 days. Carbon dioxide evolution is one of the parameters for determining the rate and extent of degradation of
any organic material. In the current study, maximum response in terms of cumulative carbon dioxide was observed in the treatment where T. reesei plus P. sajorcaju was inoculated in paddy straw (Fig. 3), thereby substantiating the above results. The pot-culture experiment (Fig. 1) showed that the application of paddy straw degraded by dual inoculation of T. reesei and P. safor-caju along with Ns0 resulted in the highest grain yield (26.7 g/pot), showing a 40% increase over N t 2o ( 19"0 g/pot). Thus, the application of paddy straw degraded with efficient fungi, in combination with Ns0 produced a significant effect on yield of wheat and partially reduced the fertilizer requirement. In general, it can be concluded that the dual culture of a cellulolytic fungus (T. reesei) and white rot fungus (P. sajor-caju), due to synergistic effects, accelerated the biodegradation of a paddy straw resulting in a nutrient-rich compost with a low C:N ratio and a high humus content which when applied to soil mineralised slowly and improved crop yield. Viesturs et al (1981) also reported that coculture of Trichoderma lignorum together with Candida lipolytica increased degradation of wheat straw.
ACKNOWLEDGEMENTS The authors are grateful to Dr K. V. B. R. Tilak, Head of the Division of Microbiology, I.A.R.I., for providing the necessary facilities.
REFERENCES
Anon. (1953). Reclamation of municipal refuse by composting. Tech. Bull. No. 9, Sanitary Engineering Research Project, University of California at Berkeley. Jackson, M. L. (1967). Soil Chemical Analysis. Prentice Hall of India, New Dehli.
188
Sarofni Kanotra, R. S. Mathur
Kahlon, S. S. & Dass, S. K. (1987). Biological conversion of paddy straw into feed. Biological Wastes, 22, 11-21. Kononova, M. M. (1966). Soil Organic Matter, Its Nature, its Role in Formation and in Soil Fertility. Pergamon, Oxford. Panse, V. G. & Sukhatme, S. V. (1978). Statistical Methods for Agricultural Workers. ICAR, New Dehli. Pramer, D. & Schmidt, E. L. (1964). Experimental Soil Microbiology. Burgess, Minneapolis, Minnesota. Viesturs, U. E., Apsite, A. F., Lankevics, T. B., Ose, V. P., Bekers, M. T. & Tengerdy, R. P. (1981). Solid state fermentation with Chaetomium cellulolyticum and Trichoderma lignorum. Biotech. Bioeng. Symp., 11,359-68.
Sarojini Kanotra Division of Microbiology, Ram Lal Anand College, Dehli University Maintained Institution, Benito Juarez Road, New Dehli- 110 021, India
& R. S. Mathur Division of Microbiology, Indian Agricultural Research Institute, New Dehli- 110 012, India (Received 5 April 1993; revised version received 16 July 1993; accepted 23 July 1993)
Short Communication
Biodegradation of Paddy Straw with Cellulolytic Fungi and its Application on Wheat Crop
METHODS The C : N ratio of chopped, air-dried paddy straw was adjusted to 60:1 by adding nitrogen as urea. Rock phosphate (1% w/w) was also added. The moisture content was adjusted to 80%. High-density polythene bags were filled with 2 kg of the material. Small holes were made throughout the bags for aeration. Before the residue was put into the bags it was inoculated with the selected four fungi singly or in combination according to the following treatment schedule:
Abstract Chopped paddy straw was used as a substrate to study the pattern of biodegradation after inoculation (singly or in combination) with four cellulolytic fungi: Trichoderma reesei QM 9414, T. viride, Pleurotus sajor-caju and Coprinus cinereus. To assess the rate of biodegradation, samples were drawn at regular intervals and analysed for organic carbon, total nitrogen, humus synthesis and respiratory activities of the degrading substrate. The C:N ratio was also calculated to judge the extent of biodegradation. Maximum lowering of the C: N ratio was observed in the treatment where dual inoculation with T. reesei and P. sajor-caju was done. A pot-culture experiment was conducted to assess the effects of application of composted paddy straw on the yield of wheat. Application of the paddy straw degraded by' the two fungi, in combination with urea at 80 kg N ha i, produced a significant effect on the yield of wheat.
( 1) (2) (3) (4) (5)
Trichoderma reesei QM 9414 Trichoderma viride 1433 Pleurotus sajor-caju Coprinus cinereus syn lagopus 3545 Trichoderma reesei QM 9414 + Pleurotus sajorcaju (6) Trichoderma reesei QM 9414 + Coprinus cinereus
(7) Trichoderma viride 1433 + Pleurotus sajor-caju (8) Trichoderma viride 1433 + Coprinus cinereus (9) Control -- uninocutated Each treatment was replicated three times. The inoculum was prepared according to the method of Kahlon and Dass (1987). The straw was inoculated with the respective inocula at 0"1% w/w (2 g/2 kg substrate). In treatments where two cultures were added, the total quantity of the inoculum (i.e. 0-1%) was kept constant and the two organisms were added in equal proportions. The temperature varied between 31"7°C and 24"8°C during the degradation period. The inoculated bags were kept in a glasshouse and sampling was done at intervals of 20, 40 and 60 days. Chemical and microbiological analyses of these samples were made to monitor the changes.
Key words': Paddy straw, cellulolytic fungi, biodegradation. INTRODUCTION The total production of rice in India is 7"5 × 107 t, which is 9"3% of the world production. Thus, approximately double this amount of paddy straw is generated every year (Economic Survey of India, 1990-1991) and disposal poses several problems. Much straw is burnt, but this has been criticised because of air pollution and respiratory problems and the danger of soil erosion due to repeated burning (Kahlon & Dass, 1987). Paddy straw could be used as an animal feed; however, it has not been popular. The major limitations as feed are low digestibility, low protein content and high lignin and silica contents. An alternative use for paddy straw could be its biodegradation to organic manure. In the present study, paddy straw was biodegraded with four cellulolytic fungi (singly or in combination) and this composted straw was applied as an organic manure to assess its effect on the yield of wheat.
Chemical and microbiological analyses The samples were dried at 60°C and ground to pass a 30 mesh (1 mm) sieve. A sample was ashed at 600°C for 4 h and the total carbon was taken to be organic matter (%)x 0"56 (Anon., 1953). Total nitrogen was determined by a Kjeldahl method (Jackson, 1967) using an autoanalyser. Humus content was estimated by the method of Kononova (1966). The rate of decomposition of paddy straw was determined by estimating the amount of carbon dioxide evolved during the process by the method of Pramer and Schmidt (1964).
Bioresource Technology 0960-8524/94/S07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain 185
186
Sarojni Kanotra, R. S. M a t h u r
Effect of biodegraded material on plant growth and yield A pot culture experiment was conducted to test the effect of application of biodegraded material prepared by cellulolytic action of selected fungi on the yield of a wheat crop (vat. HD 2285). The soil used was loamy in texture, alkaline (pH 8-5), low in organic carbon (0"38%) and total nitrogen (0"028%) contents. The quantity of available phosphorus and potash was 13"5 and 179-0 kg/h respectively. The pot-culture experiment was conducted during the winter season of 1988-1989. The temperature ranged from 6°C to 25°C during the season. The composted material prepared by the inoculation of efficient cellulolytic fungi was applied at 10 t/ha. It was mixed thoroughly with 10 kg of air-dried sieved soil. Inorganic nitrogenous fertilizer as urea was applied at 80 kg of N/ha in all the treatments except one where 120 kg N/ha was applied. A basal dressing of phosphatic
fertilizer (as single superphosphate at 60 kg P2Os/ha ) and potassic fertilizer (as muriate of potash at 40 kg P2Os/ha) was also given. A control was kept in which no fertilizer was added. The treatment schedule is seen in Fig. 1. Each treatment was replicated three times. After harvesting, plants were dried in an oven at 65°C for 72 h, and the yield of grain and straw was recorded. Statistical methods The data were analysed statistically wherever needed by using a random block design as outlined in Panse and Sukhatme (1978) and the test of significance was done at the 5% level. RESULTS AND DISCUSSION During biodegradation of paddy straw the organic carbon decreased gradually, and 10% of the control straw was lost in 60 days, while the loss was greater
55--
Nso+PADDY STRAW BIODEGRADEDBY []
50--
[] [] []
25
o ct.
[] [] []
m
v a .._I
I
15
).-
UNINOCULATED TRICHODERMA REESEI TRICHODERMA VIRIDE PLEUROTUS SAJOR-CAJU COPRINUS CINEREUS 1".REESEI -I-R SAJOR- CAJU I".REESEI-t-C.CINEREUS T.VIRIDE'F R SAJOR- CAdU T.VIRIDE-I- C, CINEREUS
Z c¢ I0 0 5
0 NO
N80 NI20
Effect of paddy straw biodegraded by cellulolytic fungi on grain yield of wheat (HD 2285).
Fig. l.
Table 1. Changes in C: N during biodegradation of paddy straw by ceilulolytic fungi Paddy straw inoculated with
Uninoculated Trichoderma reesei QM 9414 7". viride Pleurotus sajor-caju Coprinus cinereus T. reesei Q M 9414+ P. sajor-caju T. reesei QM 9414 + C. cinereus T. viride + P. safor-caju T. viride + C. cinereus
C.D. at 5% Amended C : N = 60 : 1.
Sampling time 20 Days
40 Days
60 Days
55"86 29-24 43"97 45"30 45"83 32-79 38"16 33"40 59"00
39'56 22'10 26'04 26'25 26'52 19'79 29"93 20"94 27"63
33"61 19'30 24"20 23"20 25'13 18"05 20'91 21'06 25'35
5"06
2"75
2.00
Biodegradation of paddy straw with fungi PADDY STRAW INOCULATED WITH
35-
[] [Im I~ [] [] [] [] [] []
30-
25-
o~ CO
187
20-
xxxx
UNINOCULATED TRICHODERMAREESEI TRICHODERMAVIRIOE PLEUROTUS SAOOR-CAJU COPRINUS CINEREUS T.REESEI+ P. SAJOR-CAJU T.REESEI+C.CINEREUS T.VIRIDE+ RSAJOR-CAJU T.VIRIDE+ C.CINEREUS
-r" 15 xxxx xxxx
I0 \XNN
5
0
II SINGLE INOCULATION
:ONTROL
Humus content (%) in paddy straw biodegraded by cellulolytic fungi inoculated singly or in combination.
Fig. 2.
PADDY STRAW INOCULATED WITH 2000
H UNINOCULATED 0-..-0 T.REESEI QM 9414
1800
-" "-- p. SAJOR--CAJU ,,'~ &-- -.~ T.REESEI+ -," RSAJOR/~" CAJU /
W
1600
I-
1400
//
/
I11 if)
,,~
/
1200
/
)~'
/
I000 W
0> N
0 o
800 60O 4O0 200 0
0
I0
20
30
40
50
60
70
DAYS OF INOCULATION
Fig. 3.
DUAL INOCULATION
Cumulative CO~ evolved during degradation of paddy straw inocuiated with cellulolytic fungi.
with fungal inoculation; Trichoderma reesei QM 9414 plus Pleurotus sajor-caju showed the maximum decomposition of 33%. The total nitrogen content showed an increase, the increase being maximum in the same treatment. Hence, both the fungal cultures together brought down the C:N ratio of paddy straw to 18:1 within 60 days (Table 1 ). The humus synthesis also was improved by dual inoculation of T. reesei and P. sajorcaju in comparison to the control (Fig. 2). The maximum (32"2%) humus was observed in this treatment, after an interval of 60 days. Carbon dioxide evolution is one of the parameters for determining the rate and extent of degradation of
any organic material. In the current study, maximum response in terms of cumulative carbon dioxide was observed in the treatment where T. reesei plus P. sajorcaju was inoculated in paddy straw (Fig. 3), thereby substantiating the above results. The pot-culture experiment (Fig. 1) showed that the application of paddy straw degraded by dual inoculation of T. reesei and P. safor-caju along with Ns0 resulted in the highest grain yield (26.7 g/pot), showing a 40% increase over N t 2o ( 19"0 g/pot). Thus, the application of paddy straw degraded with efficient fungi, in combination with Ns0 produced a significant effect on yield of wheat and partially reduced the fertilizer requirement. In general, it can be concluded that the dual culture of a cellulolytic fungus (T. reesei) and white rot fungus (P. sajor-caju), due to synergistic effects, accelerated the biodegradation of a paddy straw resulting in a nutrient-rich compost with a low C:N ratio and a high humus content which when applied to soil mineralised slowly and improved crop yield. Viesturs et al (1981) also reported that coculture of Trichoderma lignorum together with Candida lipolytica increased degradation of wheat straw.
ACKNOWLEDGEMENTS The authors are grateful to Dr K. V. B. R. Tilak, Head of the Division of Microbiology, I.A.R.I., for providing the necessary facilities.
REFERENCES
Anon. (1953). Reclamation of municipal refuse by composting. Tech. Bull. No. 9, Sanitary Engineering Research Project, University of California at Berkeley. Jackson, M. L. (1967). Soil Chemical Analysis. Prentice Hall of India, New Dehli.
188
Sarofni Kanotra, R. S. Mathur
Kahlon, S. S. & Dass, S. K. (1987). Biological conversion of paddy straw into feed. Biological Wastes, 22, 11-21. Kononova, M. M. (1966). Soil Organic Matter, Its Nature, its Role in Formation and in Soil Fertility. Pergamon, Oxford. Panse, V. G. & Sukhatme, S. V. (1978). Statistical Methods for Agricultural Workers. ICAR, New Dehli. Pramer, D. & Schmidt, E. L. (1964). Experimental Soil Microbiology. Burgess, Minneapolis, Minnesota. Viesturs, U. E., Apsite, A. F., Lankevics, T. B., Ose, V. P., Bekers, M. T. & Tengerdy, R. P. (1981). Solid state fermentation with Chaetomium cellulolyticum and Trichoderma lignorum. Biotech. Bioeng. Symp., 11,359-68.
Sarojini Kanotra Division of Microbiology, Ram Lal Anand College, Dehli University Maintained Institution, Benito Juarez Road, New Dehli- 110 021, India
& R. S. Mathur Division of Microbiology, Indian Agricultural Research Institute, New Dehli- 110 012, India (Received 5 April 1993; revised version received 16 July 1993; accepted 23 July 1993)