Degradation of lignocellulose by mixed cultures of cellulolytic fungi and their competitive ability

ZbI. Bakt. II. Abt, 136 (1981), 603-608 [From the Microbiology Department, Haryana Agricultural University, Hissar, India]

Degradation of Lignocellulose by Mixed Cultures of Cellulolytic Fungi and Their Competitive Ability M. M. MISHRA, K. S. YADAV and K. K. KAPOOR

Summary The changes in ejN ratio, loss in weight, degradation of cellulose and lignin in wheat straw were studied after inoculation with mixed cultures of efficient cellulolytic fungi. The loss in weight per unit time increased by inoculation with mixed cultures but there was no increase in cellulose degradation. The determination of competitive ability of these fungi indicated that Paecilomyces [ueisporus and Trichoderma viride had highest competitive ability and suppressed Trichurus epiralis, Paecilomyces varioti, Penicillium chrysogenum and Sclerotium roijsii.

Zusammenfassung Nach einer Beimpfung von Weizenstroh mit zellulolytisch effektiven Pilzen wurden Veranderung des e: N-Verhaltnisses, Gewichtsverlust, Zellulose- und Ligninabbau festgestellt. Der Gewichtsverlust pro Zeiteinheit war bei Beimpfung mit Mischkulturen erhoht, nicht hingegen der Zelluloseabbau. Paecilomyces [ueisporue und Trichoderma viride waren am konkurrenzstarksten und unterdriickten Trichurus spiralis, Paecilomyces varioti, Penicillium chrysogenum und Sclerotium rolfsii,

Many reports are available with regard to cellulolytic and lignolytio activity of microorganisms in compost and soil (BURGESS and GRIFFIN1967, CHANG and HUDSON 1967, HAIDER and DOMSCH 1969, MANGENOT 1974, MANGENOT and REISINGER 1972, REGAN and JERIS 1970, SEAL and EGGINS 1972). However, no reports are available on the use of pure cultures of microorganisms as inocula in composting, although GOLUEKE et al. (1954) used soil extract, compost extract and horse manure etc. as inocula in composting and reported that these do not enhance decomposition. Efficient cellulolytic and lignolytic fungi may enhance decomposition during composting if they can compete with native flora. The present communication reports on the degradation of wheat straw by mixed cultures of cellulolytic fungi and their competitive ability.

Materials and Methods Fungi were isolated from soil, compost heaps and organic debris:under active decomposition. The pieces of decomposing material were put directly on EGGINS and PUGH'S (1962) cellulose agar plates and incubated at 30 "C. The organisms which formed clear zones of cellulose hydrolysis were picked up and purified by subsequent transfer on cellulose agar plates. For isolation from soil, serial dilutions were poured on cellulose agar plates and incubated at 30 "C. The fungi which formed clear zones were picked and purified. To isolate fungi which may degrade lignocellulose, the decomposing pieces were added to solid medium containing wheat straw (1 mm) soaked with EGGINS and PUGH'S (1962) nutrient solution and incubated at 30 ce. There was gradual enrichment of the dominant organisms during transfer. The fungi were obtained as pure culture after many transfers. The fungi used in these studies were Paecilomyces [usisporus, Paecilomyces varioti, Trichurus epiralis, Sclerotium roljsii; Trichoderma viride 9414, Penicillium chrysogenum, Scopulariopsis sp. and Chaetomium qlobosum,

604

M. M. MISHRA et. a •

Table 1. Degradation of wheat straw by mixed cultures of mesophilic fungi (Carbon, nitrogen and C: N ratio) Mixture

Carbon (%)1)

+ T. spiralie P. [ueisporus + S. rolfsii. P. fueieporus + P. chrY80genum P. [usisporue + T. viride 9414 T. spiralis + P. chrysogenum T. spiralis + S. rolfsii S. rolf8ii + T. viride 9414 S. rolfsii + Scopulariopsie sp. P. chrysogmum + P. varioti P. [usieporus + T. spirali« P. [usisporus

Nitrogen (%) 1) Incubation time (days)

C: N ratio-)

15

30

60

15

30

60

15

30

60

52.2

51.4

47.9

0,99

1.05

1.29

52.7

49.0

37.1

51.6

49.7

49.0

0.98

1.05

1.40

52.7

47.3

35.0

51.5

50.4

49.9

0.90

0.98

1.19

57.2

51.4

41.9

51.3

50.3

49.6

0.95

0.98

1.19

54.0

51.3

41.7

52.3

49.2

47.9

1.01

1.12

1.40

51.8

43.9

34.2

52.6

51.5

49.5

0.97

1.06

1.26

53.8

48.6

39.3

51.0

49.2

47.6

0.81

0.89

1.19

63.0

55.3

40.9

50.8

50.3

49.2

0.97

1.12

1.79

52.4

44.9

27.5

51.1

48.6

48.4

1.09

1.20

1.33

46.9

40.5

36.4

50.4

49.2

48.9

1.02

1.18

1.27

49.5

41.7

38.5

S. rolfsii + P. varioti + Scopulariopsis sp.

51.3

50.3

50.3

1.15

1.26

1.43

44.6

39.9

35.2

T. spiralis + P. chrysogenum + P. oarioti

50.6

49.1

48.0

0.99

1.12

1.40

51.1

43.8

34.3

P. [usisporu« + S. rolfsii + P. chrysogenum

51.2

50.0

49.8

1.12

1.26

1.37

45.7

39.7

35.6

P. [usisporus + + C. globosum

52.8

52.3

49.8

0.78

0.91

1.12

67.7

57.5

44.5

50.7

48.6

47.4

1.12

1.26

1.39

45.3

38.6

34.1

+ S.

rolfsii

Mixture of all

r. spiralis

1) Wheat straw had 52.8 % C, 0.48 % N and a C: N ratio of 110: 1 In each of 250 ml Erlenmeyer flasks, 10 g oven dried wheat straw powder (1 mm) were taken to which 25 ml nutrient solution (MANDEI,S and STERNBERG 1976) were added. The flasks were sterilized at 15 lb. pressure for 15 min for 2 consecutive days, inoculated with a mixture of two or more fungi and incubated at 30 DC. For each treatment equal amounts of spore suspension of fungi were mixed together and 2 ml of these were inoculated. Sclerotium rolfsii which is a non-sporulating fungus was inoculated as 1 cm 2 disc, two discs being added in each flask. The loss in weight of wheat straw during degradation was determined after 15, 30 and 60 days of incubation. Each time two flasks were removed from each set and kept in oven at 105°C for 24 h. The flask with contents were weighed. The weight of contents was obtained after deducting the weight of empty flasks. The dried material so obtained was used further for determination of cellulose, lignin, carbon and nitrogen content. Cellulose and lignin in wheat straw initially and during degradation were determined by the methods of VAN SOEST and Wnm (1968). This method involved the treatment of the material with acid detergent to remove water soluble substances, fats and hem cellulose. The residue consisting of lignocellulose and ash was further treated with acetic acid buffered potassium permanganate to oxidize and solubilize lignin. It was further treated with alcoholic solutions of oxalic and hydrochloric acids to wash lignin and to remove manganese and iron oxides. Loss in weight on ashing (at 500°C) represented cellulose. The organie matter was determined by loss on ignition of 1 g of the straw at 550°C for 6 h. The car-bon values were obtained by dividing organie matter values by 1.724.

605

Degradation of Lignocellulose by Mixed Cultures

Table 2. Degradation of wheat straw by mixed cultures of mesophilic fungi (Loss in weight, cellulose and lignin degradation) Fungi

+ T. spirali« + S. roljsii P. [usisporus + P. chrysogenum P. [ueisporus + T. viride 9414 T. spirolis + P. chrysogenum T. spiralis + S. rclfsii S. rolfsii + T. viride 9414 S. roljsii + Scopulariopeis sp. P. chrysogenum + P. »orioti P. [usieporue + T. spiralis + S. roljsii S. roljsii + P. varioti + Scopulariopsis sp. T. spiralis + P. ch1-ysogenum + P. varioti P. jusisporus + S. rolfsii + P. chrysogenum P. [ueisporus + T. spirolis + C. qlobosum.

% Loss in weight

% Cellulose degradation-) Incubation tim? (days)

% Lignin degradation2 )

15

15

15

30

60

30

60

30

60

P. [ueisporus

20.0

26.3

29.5

9.2

14.4

19.0

2.1

7.2

13.1

P. jusisporus

22.1

26.3

30.0

10.7

19.5

29.8

3.7

5.6

17.2

20.0

24.7

29.5

7.1

21.3

26.4

2.3

7.4

18.1

Mixture of all

20.5

24.2

26.3

13.1

21.3

26.7

3.2

6.2

16.2

18.9

25.8

31.6

10.2

15.4

23.1

3.4

8.2

17.2

18.4

24.2

26.3

9.2

13.8

18.0

1.7

3.4

8.4

18.9

21.6

24.2

14.9

18.5

21.6

1.4

4.2

7.2

17.9

22.1

25.3

13.3

16.9

18.0

0.8

3.2

9.4

17.4

23.7

32.1

0.9

13.8

24.3

3.2

6.7

10.7

20.0

26.3

30.5

13.7

23.2

29.8

0.7

1.8

5.6

s.s

15.8

22.1

0.4

11.3

14.9

1.2

1.9

7.2

17.4

24.7

32.1

4.0

15.9

27.9

1.4

2.4

4.8

17.9

25.3

31.6

9.6

14.3

22.6

2.6

5.6

10.2

18.9

21.1

27.9

2.0

7.6

18.5

0.8

4.3

9.6

19.5

25.3

31.1

1.4

15.1

24.3

1.2

3.7

12.3

1) Cellulose in wheat straw 38.8 % 2) Lignin in wheat straw 12.6% Total nitrogen in wheat straw initially and during decomposition was determined by microKjehldah1 method (BREMNER 1969). The material was digested in sulfuric acid in the presence of potassium sulfate, copper sulfate and selenium dioxide mixture. The ammonia in the digested material was distilled in presenee of alkali and collected in 1 % boric acid solution using mixed indicator which was finally titrated with 0.02 N sulfuric acid and the nitrogen calculated. The competitive ability of different fungi was determined by the occurrence of individual fungus by plating on Martin's rose bengal agar plates, one ml of 10- 4 dilution of about 0.2 g decomposing wheat straw. Small amounts of decomposing material were put directly on the plates to allow the growth of non-sporulating or slow growing fungi. The total number of colonies of each fungus developing both on dilution plates and direct plates were counted and the percent occurrence of individual fungus was «ak-ulated.

Results and Discussion The data given in Table 1 showed that the carbon content of the decomposed material decreased only slightly although nitrogen content increased significantly. The increase in nitrogen was due to loss of carbon but the per cent carbon per g material remained almost the same. The nitrogen increase was closely related with decrease in

606

M. M. MISHRA et , al.

Table 3. Competition bet ween different mesoph ilic cellulolyt ic fung i Percentage of relative occurren ce at various da ys

Mixture

7

14

21

28

35

42

49

56

1. P . [ueisporus

100

100

100

100

100

100

100

100

2.

100 Ni l

100 N il

100 Ni l

ro 30

100 N il

100 Ni l

100 Nil

100 N il

100 Ni l

100 N il

60 40

100 Ni l

100 Nil

100 Ni l

100 N il

100 Nil

(\0 40

80 20

70 30

70 30

100 Nil

50 50

70 30

30 70

50 50

50 50

50 50

50 50

70 30

50 50

50 50

50 50

D. 100 Nil

60 40

20 80

20 80

30 70

30 70

30 70

Nil 100

Nil 100

Nil: 100

Nil 100

Nil 100

N il 100

Nil 100

Nil 100

oo 40

Nil 100

30 70

20 80

30 70

60 40

40 60

20 80

50 50

50 50

50 50

50 50

50 50

50 50

50 50

60 40

70 30 Ni l

100 N il Ni l

100 Nil Nil

100 N il N il

100 Nil N il

100 Nil Nil

100 Nil Ni l

100 Nil N il

N. D. N . D.

60 N il 40

40 N il 60

30 N il 70

60 N il 40

70 30 Nil

70 Nil 30

N il 50 50

N il 50 50

Nil 100 Nil

40 60 Nil

40 20 40

40 30 30

N il 50 50

Nil 70 30

+

80 Nil 20

90 Ni l 10

100 N il N il

100 Nil N il

100 Nil N il

100 N il N il

100 Nil N il

100 Nil Nil

+

100 Nil N il

100 Nil Nil

70 30 Nil

90 N il 10

90 Nil 10

90 Nil 10

100 Nil Nil

100 Nil Nil

+ T . sp iralis P. [usisporus + S. rolfsii

3. 1'. [usieporus

+ P.

4. P. [usisporue

+ '1'. viride

chr usoqenum

5. T. spiralis

+ P.

6. T. spirali»

+ S . rolfsii

chl'ysogenum

7. S. roljsii

+ T.

8. S. roljsii

+ Sc op u lariopsis sp.

viride

9. P. chrysogenum

+ P . uarioti

10. P . [usisporu»

P . spirali« S . roljsii

+

11. S . rolf sii + P . varioti + S copulariopsis sp , 12. T . spiralis

+

+

P. chrysogenum P. varioti 13. 1'. [usisporue S . rolfsii P . chrysogenum 14. P. fusispo rus

T . spira lis + C. qlobosu m

15. Mixtu re o f all

N.

PF50 PF50 TV70 TV7 0 TV70 TV70 TV70 TV70 TV50 TV50 PC30 TS30 TS30 PC30 PF30 PF30

GIN ratio. The decrease in GIN ra t io was maximum in combinations containing Scop ulari opsis sp. The GIN ra tio also decreased mor e in combinations containing P . chrysogenum . Higher decrease in CfN ratio with ino culation of Scopulariopsis sp. could be du e t o better assimilatio n of carbon and nitrogen in fungal mass. Our studies have shown (unpublished ) that Scopulariopsis grows on wheat straw very profusely. Th e dat a on loss in weight in Table 2 show that more t han 22 % of wheat straw was degraded in 60 days with alJ t he t rea t ments. Maximum degradation was observed in combination of T . spiralis, P. chrysogenum and P. varioti. Th e degradation was, in general, more in the treatments containing P. chrysogenum. Inoculation with

Degradation of Lignocellulose by Mixed Cultures

607

mixture of all was not better than inoculation with combinations of two or three fungi. The loss in weight was more when two or three fungi were together inoculated compared to inoculation by only one fungus, but loss in weight by mixed culture was less than the total loss in weight obtained when the fungi were inoculated separately. This was due to the fact that there was competition in the mixed culture and therefore all were not able to grow equally. The loss in cellulose during degradation of wheat straw varied from 18 to 29 % and the maximum loss in cellulose was with P. [usisporus and S. roljsii (Table 2). Comparison of cellulose degradation by single and mixed cultures (KAPOOR et. al. 1978, MISHRA et. al. 1979) showed that cellulose degradation did not increase by use of mixed cultures, In single culture P. [usisporus degraded 25 % and S. roljsii 40 % cellulose in 60 days. But when S. roljsii was used with other fungi, degradation of cellulose was always less. The degradation of lignin was less as compared to celluose. There was no linear relationship between cellulose and lignin degradation. The examination of relative occurrence of different fungi revealed that P.jusisporus had the highest competitive ability (Table 3). Trichoderma viride could grow alongwith P. [usisporus, but other fungi like T. spiralis, S. roljsii and P. chrysogenum were completely s.uppressed by P. [usisporus. The competitive ability to T. spiralis, P. chrysogenum, P. varioti and S. roljsii were equal. Less degradation of cellulose in combinations containing S. roljsii as compared to S. roljsii alone, was due to competition by other fungi. The results indicated that P. [usisporus and T. viride because of higher competitive ability appear to be most suitable for use as inocula in composting

Acknowledgements The authors are thankful to Indian Council of Agricultural Research, New Delhi, for financial support and Dr. P. TAFRO, Professor of Microbiology, for suggestions.

References BREMNER, J. M.: Inorganic forms of nitrogen. In: Methods of Soil analysis: Chemical and microbiological properties. Vol. 2. (C. A. BLACKet al., eds.], American Society of Agronomy, Madison, Wisconsin, 1965. BURGESS, W., and GRIFFIN, D. M.: Competitive saprophytic colonization of wheat straw. Ann. Appl, BioI. 60 (1967),137 -142. CHANG, Y., and HUDSON, H. J.: The fungi of wheat straw compost. 1. Ecological studies. Trans. Brit. Mycol. Soc. 50 (1967),649-666. EGGINS, H. O. W., and PUGH, G. J. F.: Isolation of cellulose decomposing fungi from soil. Nature (Lond.] 193 (1962), 94-95. Gor,uEKE, C. G., CARD, B. J., and MCGAUHEY,P. H.: A critical evaluation of inoculum in eornpoa> ing. Appl, Mierobiol. 2 (1954), 45-53. HAIDER, K., und DOMSCH, K. H.: Abbau und Umsetzung von lignifiziertem Pflanzenmaterial durch mikroskopische Bodenpilze. Arch. Mikrobiol. 64 (1969), 338-348. KAPOOR, K. K., JAIK, M. K. MISHRA, M. M., and SINGH, C. P.: Cellulase activity degradation of cellulose and lignin and humus formation by cellulolytic fungi. Ann. Microbiol. (lnst. Pasteur) 129B (1978), fil3 -620. MANDELS, M., and STERNBERG, D.: Recent advances in cellulase technology. J. Ferm. Technol. 54 (1976), 267 - 286. MANGENOT, F.: New data concerning lignolytic activity of soils. Ann. Microbiol. Enzimol. 24 (1974), 47 -50. ~ and REISINGER, 0.: Lignolytie activity in soils. In: Proceedings of the Symposium on Soil Microbiology. Symp. bioI. Hung. 11 (1972), 147 -152. MISHRA, M. M., SINGH, C. P., KAPOOR, K. K., and JAIN, M. K.: Degradation of lignocellulosic material and humus formation by fungi. Ann. Mierobiol. (Inst. Pasteur) 130A (1979), 481486.

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11. M. MISllRAet. al., Degradation of LignoceJlulose by Mixed Cultures

REGAN, R . 'V., a nd JERIS, 1. S. : A review of decomposition of ceJlulose an d refuse. Compost. Sci. 11 (1970), 17 - 20. SEAL, K. J., an d EG GINs, H . O. 'V.: The role of mi cro-organisms in the biodegrada tion of farm animal wa stes with particular refer en ce to intensi vel y produced wastes. A review. Int. B iod etn. Bull. 8 (1973), 95 - 100. VAN SOEST, P . J., an d WINE, R . H. : Determination of lign in an d ceJlulose in ac id detergent fib er with per m anga nat e. J . Asso c. Off . An al. Chern. 51 (1968) , 782 - 785. Authors' address : M. M. MISHRA, K . S. Y ADA V an d K. K. KApo OR, D epartment of Microbiology, H aryana Agri· cu lt ur al Universit y, Hi ssar - 125004, India .