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CELLULOSE-DESTRUCTING SOIL MICROMYCETES OF UZBEKISTAN AND INFLUENCE OF SOME FACTORS ON CELLULOLYTIC ACTIVITY..,
Mukhammadiev Bakhtiyor Kurbanmuratovich, Tashkent State Agrarian University, University, Tashkent, Uzbekistan E-mail: [email protected] Djumaniyazova Gulnara Ismailovna, Academy of Sciences Republic of Uzbekistan Institute of Microbiology, Tashkent, Uzbekistan E-mail: gulnara [email protected]
CELLULOSE-DESTRUCTING SOIL MICROMYCETES OF UZBEKISTAN AND INFLUENCE OF SOME FACTORS ON CELLULOLYTIC ACTIVITY AND SACCHARIFYING ABILITY OF TRICHODERMA HARZIANUM
Abstract: Cellulose degradation by soil fungi can provide with valuable products such as sugar and proteins that can be used in husbandry or food industries. 1897 strains of soil fungi of 146 species belonging to 35 genera and four subkingdoms have been evaluated for cellulose degrading activity and sugar and protein synthesis. 45 species have expressed good abilities for that, and Trichoderma harzianum was the most active among them. Various plant raw materials (filter paper, residues of camel thorn, cotton leaves, corn stumps, wheat straw, rice straw and rise husks) have been evaluated as media for cultivation of T. harzianum, and the most appropriate one recognized was camel thorn residues. Dynamics ofboth cellulose degradation and saccharification of the substrata by the fungus has been determined; optimum temperature and reaction of media for growth of T. harzianum and maximum production of sugars have been identified.
Keywords: soil, micromycetes, enzymes, cellulose, cellulolytic activity, mineralization, proteins, biosynthesis, hydrolysis, hydrolyzate, sugars, growing microorganisms.
1. Introduction Metabolic processes in microorganisms, biosynthesis of
Increasing crop's yields is dependent on improving of biologically active substances and cellulase in particular
metabolism in plants, and the latter, in its turn, is signifi- are influenced by growing conditions and content of nu-
cantly conditioned by the mineralizing of plant residues tritional media [2]. Various organic and mineral sources
in the soil. These residues mainly consisted of cellulose. of nitrogen can be used for growing microorganisms that
Ability to degrade it is found in many species of fungi, are cellulase producers [3].
bacteria, including thallobacteria (formerly actinomy- Purpose of our investigations was to identify species
cetes). Having miscellaneous enzymes soil micromycetes composition of soil micromycetes that are active produc-
can use various substrata as a food, and this could be one ers of cellulose-degrading enzymes and proteins. We
of reasons of their wide occurrence in natural habitats have studied some features of cellulase biosynthesis by
[1]. There are plenty of data in the scientific literature on Trichoderma harzianum Rifai and its saccharifying ability.
distribution of cellulose-degrading fungi that live in vari- 2. Materials and methods
ous soil types and in different numbers [5]. In Uzbeki- Common mycological methods of isolation of mistan cellulose-degrading soil micromycetes have been a cromycetes have been used in our investigations.Cellu-subject of investigations carried out by M. Sh. Sagdullae- lolytic activity of fungi has been studied in laboratory va [7] a.o. At the same time many ofthese investigations and field conditions in accordance with methods de-were occasional as a part of studies of soil mycobiotas. scribed by A. A. Imshenetzky. Their ability to degrade
Section 10. Agriculture
cellulose was determined by the filter paper method in the Mandel's medium [4]. Trichoderma harzianum has been grown in 250 ml Erlenmeyer flasks containing 50 ml of Mandel's nutrition broth with pH 5.5, on shakers, using a method of the submerged cultivation, during 72 to 96 hours, at 30oC. Cellulase activity of Trichoderma harzianum has been determined on the base of increasing concentration of sugars in the reaction mixture after hydrolysis of the substrate (cotton lint overgrown with fungal mycelium) comparing with the check treatment (cotton lint without mycelium) [1].
Saccharifying ability of fungi has been determined on the base of production of reducing sugars under influence of enzymes of the culture fluid in hydrolyzates of plant residues after Somogyi [6].
3. Results and Discussion
1897 strains of micromycetes belonging to 146 species, 35 genera and four subkingdoms of fungi have been isolated in total from three soil types, namely old-arable soils irrigated during long times (i), and soils that were recently introduced in agriculture (ii), and typical sero-zem soils (iii). From these 45species of the genera Aspergillus, Chaetomium (anamorph stage Botryotrichum),
Fusarium, Penicillium and Trichoderma have had a notable ability to degrade cellulose. Table 1 shows dynamics of cellulose biosynthesis by one of these species, namely Trichoderma harzianumThe following species have been selected for further and more detailed investigations as the most active producers of cellulolytic enzymes and proteins: Alternaria alternate (Fr.) Keissl. (syn. A. tenuis Nees), Aspergillus flavus Lk., Cladosporium herbarum Lk., Fusarium sp. (as F. moniliformes.l., nom. ill.), Trichoderma harzianum and T. viride (Fr.) Pers. (syn. T. lignorum Harz.). Among these species Trichoderma harzianum has had the highest ability to degrade cellulose and it has been used in further investigations.Results of investigations on determining of cellulose biosynthesis dynamics on two media, namely pure cellulose (filter paper) and plant residues (camel thorn - Alhagicamelorum Fisch.) have shown that intensity of cellulase accumulation in the culture fluid has been the most intensive during first 3 to 4 days of cultivation, then intensity of the process has decreased. Such a decrease of biosynthesis intensity has been noted in both of substrates; however cellulolytic activity of the fungus has been slightly higher on camel thorn residues comparing with filter paper (Table 1).
Table 1.- Dynamics of cellulose biosynthesis by Trichoderma harzianum
Material containing cellulose Cellulolytic activity, unit/ml, after cultivation during hours
24 48 72 96 120 144
Filter paper 0.10 0.20 0.50 0.70 0.76 0.84
Camel thorn 0.20 0.40 0.63 0.81 0.90 0.97
It has been found that slightly acidic or neutral reaction of the original nutrition medium has favoured synthesis of cellulase. Activity of enzymes has been on the same level between pH 5.0 and pH 7.0. Decreasing pH till 4.5 has affected negatively on growth and activity of the producer-fungus, and its growth has stopped at pH 4.0. It has been supposed that various types of this enzyme with different pH optimums have existed in the camel thorn medium while maximum activity on the filter paper medium has been observed at pH 5.5. Culture of the fungus has revealed sensitivity to the temperature during cultivation. So, the least biosynthetic activity and weak growth have been registered at 25oC to 27oC, and increasing it till 30oC to 32oC has raised biosynthetic activity of the fungus 2-3 times and fungal growth has been profuse as a dense suspension. Modifications of Mandel's medium have been tested as well, where one
of its ingredients has been excluded in each of two media used in our investigations. Elimination from the media of the most important ingredient - source of the cellulose in the form of the filter paper or plant residue, has led to zero-activity of the cellulolytic activity. Then, replacing of the source of cellulose with easy for assimilation product as glucose has led to the suppression of biosynthesis. This apparently can evidence that process ofbiosynthesis of cellulase by Trichoderma harzianum has an inducible character. Further, elimination from the medium of the nitrogen source in the form of (NH2)2HPO4 has resulted in decreasing cellulolytic activity 2-3 times, comparing with a full-medium. Other mineral ingredients of the medium have effected on the fungal activity variously depending on the source of celluloseThe highest cellulolytic activity on the 3rd day of cultivation has been determined, except camel thorn residues, on wheat straw. So,
CELLULOSE-DESTRUCTING SOIL MICROMYCETES OF UZBEKISTAN AND INFLUENCE OF SOME FACTORS ON CELLULOLYTIC ACTIVITY...
it has been found, that, in relation to needs of production, inexpensive and simple nutrition media containing various sources of cellulose can be used with this purpose. Further investigations have been carried out for evaluation of saccharifying ability of Trichoderma harzianum on different waste materials containing cellulose. Firstly, substrata have been cut using knife-mill into pieces with length 0.2 to 0.5 mm. Then these dry materials have been undergone to processing with an active culture fluid of the fungus with pH 5.5 in ratio 1:15. It has been cultivated in shaker at 180-200 rpm during six hours at 30oC,
and then content of sugars in hydrolyzate has been de-termined.Results have shown that the highest concentrations of sugars have been registered after enzymatic hydrolysis of the camel thorn residues and corn stumps (Table 2). Evidently, this can be explained by the availability of these products for cellulases and xylanases of Trichoderma harzianum.Degrees of conversion of these products have been 30% to 50%. This parameter has been much lesser for rice husks (10%), apparently because of solid association of these crystalline polymers with hemi-cellulose and/or lignin.
Table 2.- Dynamics of saccharification of cellulose-containing raw materials by cellulolytic enzymes of Trichoderma harzianum
Material containing cellulose (plant residues) Amount of sugars (mg/ml) in hydrolyzates, after cultivation during hours
1 2 3 4 5 6
Cotton leaves 1.5 1.7 2.4 2.7 2.6 2.4
Corn stumps 2.3 2.8 3.04 3.2 3.0 2.7
Wheat straw 1.86 2.3 2.7 2.9 3.0 2.7
Camel thorn 2.4 2.8 3.4 3.6 3.7 3.4
Rice straw 1.3 1.7 1.9 2.1 2.4 2.2
Rise husks 0.4 0.6 0.7 0.9 1.1 1.0
Hydrolysis of cellulose-containing substrata has to slowing of the cellulolytic action of the culture fluid
been the most intensive during the first hours of the and, respectively, to decrease of producing sugars.
trial. Then, decreasing amount of the enzyme has led
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