Isolation and identification of some bacterial and fungal isolates based on qualitative test of cellulase and ligninase activity from different sources

The existence of agricultural wastes, especially wood and degradation-resistant wastes and time-consuming composting of these residues, shows the importance of using degrading microorganisms as bioactivators. By inoculating microorganisms into hard-to-decompose agricultural residues, it is possible to increase their composting rate and produce quality compost. The aim of this study was to isolate, identify and determine the efficacy of cellulolytic and ligninolytic bacteria and fungi.

Samples were taken from semi-rotten tissues of trees, organic matter including compost, vermicompost, animal manure, animal samples such as termites, earthworms, amur fish and commercial samples containing bioactivators. In order to isolate bacterial and fungal isolates with the ability to produce cellulose and ligninase enzymes, after preparing a series of dilutions from the extracts of each sample, culture was performed on common culture media. After purification of bacteria and fungi, the efficiency in terms of cellulase and ligninase activity was determined by measuring the diameter of the transparent halo on specific culture media. Molecular identification of selected bacterial and fungal isolates was performed by amplification of 16S rDNA region and ITS1-5.8S-ITS2 region, respectively.

A total of 83 bacterial isolates and 18 fungal isolates capable of producing cellulase were identified. BB12 and CCB9 isolates showed the highest cellulase activity with a ratio of halo diameter to colony diameter of 9.5 and 7.5, respectively. In fungal isolates, the highest cellulolytic activity was related to WF2 and WF4 isolates; the ratio of halo diameter to colony diameter in each of these two isolates was 4.57 and 2.4, respectively. Regarding ligninolytic activity, it was found that Chapak culture medium containing one gram of methyl blue is the best medium to study ligninolytic activity and the highest ratio of halo diameter to colony diameter in Chapak medium was in WB6 and WB5 bacterial isolates, respectively in the amount of 10 and 7. In fungal isolates, ligninolytic activity was observed only in WF2 and WF4 isolates; So that the ratio of halo diameter to colony diameter was 2.17 and 2, respectively. The results of molecular identification showed that the selected bacterial isolates were most similar to Bacillus halotolerans, Bacillus siamensis, Bacillus paralicheniformis, Bacillus subtilis, Bacillus atrophaeus, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus sercatus, Bacillus sp. The fungi were more similar to Penicillium corylophilum and Pseudogymnoascus pannorum. The maximum parsimony tree with 1000 bootstrap replications showed that bacterial and fungal isolates were located in 9 and 2 clades, respectively.

Examination of cellulolytic activity showed that Bacillus amyloliquefaciens showed the highest amount of cellulolytic activity and the highest ligninolytic activity was seen in Serratia marcescens. In fungal isolates, the highest cellulolytic and ligninolytic activities were observed in Penicillium corylophilum and Pseudogymnoascus pannorum. Based on the results of this study, further investigation is suggested about the possibility of using a combination of selected microorganisms to prepare initial formulations for faster conversion of agricultural waste into compost.