Bioconversion of straw into improved fodder: fungal flora decomposing rice straw.

The fungal flora decomposing rice straw were investigated all over the soil of Sharkia Province, east of Nile Delta, Egypt, using the nylon net bag technique. Sixty-four straw-decomposing species belonging to 30 genera were isolated by the dilution plate method in ground rice straw-Czapek's agar medium at pH 6. The plates were incubated separately at 5℃, 25℃ and 45℃, respectively. Twenty nine species belonging to 14 genera were isolated at 5℃. The most frequent genus was Penicillium (seven species), and the next frequent genera were Acremonium (three species), Fusarium (three species), Alternaria, Chaetomium, Cladosporium, Mucor, Stachybotrys (two species) and Rhizopus stolonifer. At 25℃, 47 species belonging to 24 genera were isolated. The most frequent genus was Aspergillus (nine species), and the next frequent genera were ranked by Penicillium (five species), Chaetomium (three species), Fusarium (three species). Each of Alternaria, Cladosporium, Mucor, Myrothecium and Trichoderma was represented by two species. At 45℃, 15 species belonging to seven genera were isolated. These were seven species of Aspergillus, two species of Chaetomium and two species of Emericella, while Humicola, Malbranchea, Rhizomucor and Talaromyces were represented by one species respectively. The total counts of fungi the genera, and species per gram of dry straw were significantly affected by incubation temperature and soil analysis (P < 0.05).

Rice (Oryza sativa L), one of the world's leading crops, is cultivated in about 2 × 106 feddans in Egypt and the production of rice straw reaches about 8 × 106 tons per year. Straw is usually either burnt in the field causing environmental hazards such as respiratory diseases or disposed in a way that does not benefit the farmers to its maximum extent (Arai et al., 1998; Samar et al., 1999; Torigoe et al., 2000). Fungi are the most important group among microbial agents for straw decomposition (Hudson, 1972; Srinivason, 1979; Harper and Lynch, 1982a; Yananobe et al., 1994; Morais et al., 1999; Tengerdy and Szakacs, 2003). There have been many surveys of cellulose-decomposing fungi but most fungi were isolated directly from soil or other sources on pure cellulose (Abdel-Hafez et al., 1978; Abdel-Hafez and Abdel-Kader, 1980; Mazen et al., 1980; Abdel-Hafez, 1982; Abdel-Kader et al., 1983; Moubasher et al., 1985). Surveys on wheat straw and other cultural wastes were conducted by other workers (El-Nawawy, 1972; El-Kady et al., 1981; Abdel-Hafez et al., 1990). Moubasher et al. (1985) isolated 19 species (one variety) belonging to 13 genera from wheat and broad bean straw compost at 45℃. However, few studies were focused on rice straw. Coronel et al. (1991) found that Aspergillus fumigatus was the most active cellulase producer on rice straw substrate among 144 tested strains of thermophilic lignocellulose-degrading fungi. Rai et al. (2001) isolated 42 mesophilic species from litter of rice straw.

Research on the bioconversion of these agricultural residues into a microbial biomass as an improved feed supplement has been conducted in this study. The present investigation aimed the mycoflora inhabiting rice straw at 5°, 25° and 45℃ in different localities.

Materials and Methods

Samples and soil analysis

Nylon net bag technique (House and Stinner, 1987; Wise and Schaefer, 1994) was used in this study. Thirty nylon bags each containing 10g of rice straw chopped to lengths of 5~6 cm, were prepared. These bags were distributed in different places throughout 10 areas (three bags in each area) of Sharkia Province of Egypt, in which large quantities of rice straw are produced. These sampling areas were; Abo-Kabir, Diarb-Negm, Hehia, Mashtool, Menia El-Kamh and Zagazig soils where were cultivated with Triticum vulgare and Trifolium alexandrinum after harvesting of paddy rice, Oryza sativa. Abo-Hamad and Fakoos areas were Mangifera indica orchards while Belbeis and Kafr-Sakr were orchards of Citrus sinensis. The bags were buried 5~10 cm from the soil surface for 10 days, then collected again for investigation.

Soil samples collected from the same localities at which nylon net bags were buried, were analyzed chemically for total soluble salts and organic matter content (Jackson, 1962). The soil texture was determined by the sieve method (Piper, 1947) using a standard, Rot-Top electric sieve shaker, VEB Metall Weberei Neustadi, Orta D.D.R. The reaction of the soil was measured using the glass electrode pH-mater (Richards, 1954).

Estimation of cellulose decomposing fungi

Fungi inhabiting rice straw were estimated using dilution plate method as described by Johnson et al. (1959). One ml of the straw suspension was introduced to each agar plate using Menzies dipper (1957) as recommended by Watson (1960) and Moubasher (1963). Modified Czapek's agar medium was used in which ground rice straw replaced sucrose (20 g/l) and to which rose Bengal (1/15000) was added as a bacteriostatic agent (Smith and Dawson, 1944), and the pH of the medium was adjusted to 6.0. Fifteen plates for each bag were used and incubated at 5°, 25° and 45℃ (five plates at each temperature degree) for 15 days. The plates were examined daily. The average number of colonies per dish was multiplied by the dilution factor to obtain the number of colonies/g dry straw in the original rice straw sample.

Fungal identification

The fungal colonies appeared were picked up on Czapek's 0.5% yeast extract, malt extract and/or potato dextrose agar media, purified using spore technique and identified in this laboratory by consulting the following works: Gams et al., 1998; Kubicek and Harman, 1998; Moubasher, 1993; Kitch and Pitt, 1992; Pitt, 1986; Domsch et al., 1980; Pitt, 1979; Booth, 1977; Raper and Fennell, 1977; Ellis, 1971; Rifai, 1969; Raper and Thom, 1968 ; Cooney and Emerson, 1964.

Statistical analysis

The obtained data were conducted to one-way ANOVA and multiple-way ANOVA by Snedecor and Cochran (1982) and differences between means were done at the 5% probability level using Duncan's new multiple range tests (Duncan, 1955). Bivariate correlation matrix of the obtained data was done using by SPSS software program (ver. 8) as described by Dytham (1999).

Results and Discussion

The straw dilutions (of each nylon net bag) used for estimation of rice straw-degrading fungi were 10-4 in the case of incubating the plates at 5° and 45℃, and 10-5 for 25℃. Rai et al. (2001) used dilution 10-4 for isolation of litter decomposing mycoflora of rice straw using nylon net bag technique. They also found that dilution plate method yielded maximum number of fungi in comparison with direct observation and damp chamber incubation methods.

Results in Tables 1 and 2 show that the total count of fungi per gram of dry straw was significantly affected (P < 0.05) by incubation temperature, pH value (-ve r), organic matter (+ve r) and total soluble salts (-ve r) of the soil where the nylon net bag was buried. The fungal-rich soils were sample no. 10, 9, 8, 4, 6 and 2, which are characterized by clay type, to be slightly acidic to neutral (6.9~7.2 pH-value) with high organic matter content (1.87~1.53%), and low total soluble salts (0.46~0.63%), and cultivated with Triticum and Trifolium. However the fungal-poor samples were soil sample no. 3 and 5 which are sandy, alkaline (7.8 and 7.7 pH), with low organic matter content (0.25 and 0.41%), high in total soluble salts (2.20 and 1.67%) and cultivated with Citrus and Mangifera. Abdel-Hafez (1978) and Helal (1993) found that the total population of fungi was influenced by the content of the total soluble salts of the soil. The highest fungal population was recorded from sample no. 10 (Zagazig area) which showed, 7.2 × 104, 7.1 × 105 and 5.8 × 104 colony/g dry straw, while the lowest fungal population with 1.4 × 104, 7.8 × 104 and 1.7 × 104 colony/g dry straw from sample number 3 (Belbeis area) at incubation temperature 5°, 25° and 45℃, respectively. Abdel-Sater and El-Said (2001) identified twenty three species belonging to 11 genera at 28℃ from 30 samples of rice straw from Qena Governorate (South Valley), Egypt with total count 7.3 × 103 colonies/mg.

Table 1

Characterization of soil samples tested and rice straw-decomposing fungi

1-The same letter in the same column/row means not significant at p < 0.05.

2-a, b, c, ... for column comparison and x, y, z for row comparison.

Table 2

Pearson correlation matrix (r) and probability showing the relationship between soil analyses (pH, OM and TSS) and no. of genera, no. of species and total count at different incubation temperatures

*Correlation is significant at the 0.01 level.

**Correlation is significant at the 0.001 level.

Sixty-four species belonging to thirteen genera were collected in the present investigation (Table 3). The most diverse fungal flora was obtained at 25℃ (24 genera and 47 species) with total fungal count 4.3 × 105 colony/g dry straw, followed by 14 genera and 29 species at 5℃ with 3.6 × 104 colony/g dry straw, while only 7 genera and 15 species isolated at 45℃ with the total fungal count 4.1 × 104 colony/g dry straw. Most of the genera which appeared at 5℃ were also occurred on 25℃ except Botrytis, Oidiodendron and Pestalotia. These genera can grow at 25℃ (Helal, 2005) but their slow growth may be prevented in the presence of rapidly growing mesophilic species. Results in Tables 3 and 4 also indicated that at 45℃ the following genera appeared: Chaetomium thermophilum, Humicola grisea var thermoidea (Scytalidium thermophilum), Malbranchea sulfurea (M. cinnamomea), Rhizomucor pusillus and Talaromyces thermophilus. They are thermophilic, while seven species of Aspergillus, the two isolates of Emericella nidulans (Aspergillus nidulellus) and Chaetomium spirale appeared at both 25° and 45℃ i.e. they are thermotolerant. These two groups were previously described by Domsch et al. (1980), Moubasher (1993) and Mouchacca (1997 and 2000a and b).

Table 3

Characterization of rice straw-decomposing fungi isolated at 5°, 25° and 45℃ in rice straw-Czapek's agar medium

NS : number of species, NCI : number of cases of isolation, OR : occurrence remarks, TC : total count, TNS : total number of species.

H = high occurrence isolated more than is 15 cases (out of 30).

M = moderate occurrence from 8 to 15 cases.

L = low occurrence from 4 to 7 cases.

R = rare occurrence, less than 4 cases.

Table 4

Fungi recovered in ground rice straw-Czapek's agar at 5°, 25° and 45℃ in 30 rice straw samples using Nylon net bag technique and dilution plate method

TC : total count, NCI : number of cases of isolation, OR : occurrence remark.

H = high occurrence isolated more than is 15 cases (out of 30).

M = moderate occurrence from 8 to 15 cases.

L = low occurrence from 4 to 7 cases.

R = rare occurrence, less than 4 cases.

As shown in Table 4 the most frequent genus which appeared during this study was; Aspergillus (10 species), and the next frequent fungal genera were ranked by Penicillium (9 species), Chaetomium and Fusarium (4 species), Acremonium (3 species), Alternaria, Cladosporium, Cochliobolus, Emericella, Humicola, Mucor, Myrothecium, Stachybotrys and Trichoderma (2 species). Members of Aspergillus, Penicillium and Trichoderma were also found to be the most prevalent fungi isolated from rice straw as xylan decomposing fungi (Abdel-Sater and El-Said, 2001).

Aspergillus represented 100% of the samples at 25° and 45℃, constituting 33.4% and 60.4% of total fungi, respectively, but did not appear at 5℃. A. awamori, A. flauvs, A. fumigatus, A. niger and A. terreus were of high occurrence at both 25℃ and 45℃, while A. flavus var columinaris was of low occurrence at both temperatures. At 25℃ A. ochraceus and A. sydowii were of moderate occurrence, while A. tamarii was of rare occurrence. At 45℃ A. ustus was of rare occurrence and represented 10% of the samples, and can grow at 25℃ (Helal, 2005). The six species isolated at 45℃ were thermotolerant fungi as mentioned above. These ten species of Aspergillus were isolated previously on cellulose medium from different sources (El-Kady et al., 1981; Abdel-Kader, 1983; Abdel-Halez et al., 1990; Rai et al., 2001).

Penicillium was the second most common genus isolated at 5℃ and 25℃, but not at 45℃, and represented 100% and 83.3% of the samples constituting 15.4 and 8.3% of the total count of fungi, respectively. P. citrinum, P. oxalicum were of high occurrence and P. corylophilum was of low and moderate occurrence at both 5℃ and 25℃, respectively. On the other hand, P. canescens was of low occurrence, P. chrysogenum was of high occurrence, P. hrequei was of rare occurrence, and P. verrucosum was of moderate occurrence at 5℃, while P. janthinellum and P. rubrum appeared only at 25℃ with rare occurrence. Penicillium species were also isolated frequently from agricultural residue, compost and manures in Egypt (El-Dohlob et al., 1985) and other parts of the world (Satyanarayana et al., 1988; Banerjee et al., 1995).

Chaetomium came third after Aspergillus and Penicillium. It was isolated at 5°, 25° and 45℃ and represented 90, 73 and 73% of the samples constituting 8.4, 3.9 and 4.8% of total count of fungi, respectively. C. cochliodes was of moderate occurrence and C. globosum was of high occurrence at 5° and 25℃, C. spirale was of moderate occurrence at 25° and 45°, while C. thermophilum, a thermophilic fungus, occurred only at 45℃. Soytong (1991) found that some Chaetomium species isolated from the rhizosphere of some economical plants can degrade rice husks, rice straw and paper.

Fusarium occupied the fourth place was isolated at 5° and 25℃ but did not appear at 45℃ and represented 96.7 and 86.7% at both temperatures constituting 7.8 and 8.0% of the total count of fungi, respectively. F. moniliforme was of moderate occurrence and F. solani was of high occurrence at both 5° and 25℃. F. oxysporum was of high occurrence at 25℃ only, while F. pallidoroseum (F. semitectum) was of high occurrence at 5℃. El-Kady et al., (1981) isolated F. oxysporum and F. moniliforme in high occurrence on celluose medium from wheat straw. Also, Rai et al. (2001) reported that F. semitectum was dominant on rice straw.

Acremonium consisted of three species. A. fusidioides was of moderate occurrence and A. kiliense was of high occurrence only at 5℃. A. strictum appeared at 5 and 25℃ in low and rare occurrence, respectively. Acremonium represented 76.7 and 10% of the samples constituting 7.8 and 0.3 of the total count at 5° and 25℃.

Cladosporium was isolated only at 5° and 25℃, and represented 100% and 83.3% of the samples constituting 17.6 and 7.1 of the total count of fungi. C. cladosporioids was of high occurrence at 5℃ and moderate occurrence at 25℃, while C. herbarum was of high occurrence at both temperatures.

Cochliobolus appeared only at 25℃, represented in 36.7% of the samples constituting 1.5% of the total count. C. lunatus was of moderate occurrence,while C. sativus was of low occurrence.

Humicola appeared at 25° and 45℃ represented 30 and 43.3% of the samples, constituting 0.9 and 1.5% of the total count of fungi. H. grisea Traaen was of moderate occurrence at 25℃ only, while H. grisea var thermoidea (Scytalidium thermophilum) was of moderate occurrence only at 45℃.

Emericella nidulans (Aspergillus nidulellus) was isolated at 25° and 45℃ and represented 43.3 and 53.3% of the samples constituting 1.5 and 3.1% of the total fungi. E. nidulans var. Lata was of moderate occurrence at 25 and 45℃ and E. quadrilineata occurred low at both temperatures.

Mucor was isolated at 5° and 25℃, represented 93 and 86.7% of the samples constituting 7.3 and 6.8% of the total fungi. M. circinelloides was of moderate occurrence and M. racemosus was of high occurrence at both temperatures.

Myrothecium was isolated only at 25℃, represented 73.3% of the samples constituting 5.8 of the total fungi. M. roridum was of high occurrence, while M. verrucaria was of low occurrence.

Stachybotrys was isolated at 5° and 25℃, represented 100% and 43.3% of the samples constituting 14.9 and 1.3% of the total count of fungi. S. chartarum was of high occurrence at 5℃ and moderate occurrence at 25℃, while S. elegans was of high occurrence only at 5℃.

Trichoderma appeared only at 25℃ and represented 86.7% of the samples constituting 8.5% of the total fungi. This genus consisted of two species T. harzianum and T. koningii which were of high occurrence. Trichoderma species were often cited as high cellulose-decomposers (Moharram et al., 1995; Tengerdy and Szakacs, 2003; Zayed and Abdel-Motaal, 2005).

The other genera that appeared in this study represented one species. Botrytis cinerea, Oidiodendron griseum and Pestalotia sp. appeared only at 5℃. Botrytrichum piluliferum, Colletotrichum dematium, Gliocladium catenulatum, Phoma herburum, Scopulariopsis brevicaulis, Trichothecium roseum and Verticillium catenulatum appeared only at 25℃. Malbranchea sulfurea, Rhizomucor pusillus and Talaromyces thermophilus appeared only at 45℃. Epicoccum nigrum, Nigrospora sphaerica and Rhizopus stolonifer appeared at 5° and 25℃. No species appeared at the three incubation temperatures, 5°, 25° and 45℃. Most of these fungi isolated on rice straw during this study were isolated with different frequency of occurrence from various hemicellulosic and cellulosic materials in Egypt (Moubasher and Mazan, 1990; Moubasher, 1993; Abdel-Sater and El-Said, 2001) as well as other parts of the world (Caldwell, 1973, Bisaria and Ghose, 1981; Maheshwari et al., 2000).