Design and application of an efficient cellulose-degrading microbial consortium and carboxymethyl cellulase production optimization

Abstract

Microbial consortia with high cellulase activities can speed up the composting of agricultural wastes with high cellulose contents and promote the beneficial utilization of agricultural wastes. In this paper, rabbit feces and sesame oil cake were used as feedstocks for compost production. Cellulose-degrading microbial strains were isolated from compost samples taken at the different composting stages and screened via Congo red staining and filter paper degradation test. Seven strains, Trichoderma reesei, Escherichia fergusonii, Proteus vulgaris, Aspergillus glaucus, Bacillus mycoides, Corynebacterium glutamicum, and Serratia marcescens, with high activities of carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), and β-glucosidase (β-Gase) were identified and selected for consortium design. Six microbial consortia were designed with these strains. Compared with the other five consortia, consortium VI composed of all seven strains displayed the highest cellulase activities, 141.89, 104.56, and 131.18 U/ml of CMCase, FPase, and β-Gase, respectively. The single factor approach and response surface method were employed to optimize CMCase production of consortium VI. The optimized conditions were: culture time 4.25 days, culture temperature 35.5°C, pH 6.6, and inoculum volume 5% (v/v). Under these optimized conditions, the CMCase activity of consortium VI was up to 170.83 U/ml. Fermentation experiment of rabbit feces was carried out by using the consortium VI cultured under the optimal conditions. It was found that the application effect was better than other treatments, and the fermentation efficiency and nutrient content of the pile were significantly improved. This study provides a basis for the design of microbial consortia for the composting of agricultural wastes with high cellulose contents and provides a support for beneficial utilization of agricultural wastes.

Keywords: carboxymethyl cellulase; cellulose degrading strains; condition optimization; isolation; microbial consortium; rabbit feces; sesame oil cake.

FIGURE 1

Schematic diagram showing the layout of strain inoculation on plates in the antagonism test. In this figure, A represents a random dominant strain and BCDEF, etc. represent other different dominant strains. After streaking on the plate, we observed whether there was a weak or non-growing antagonistic phenomenon at the intersection of A strain and BCDEF, etc. strain, and if it grew well, there was no antagonistic effect. Further, we observed whether strain B and strain CDEF, etc. were antagonistic, and so on.

FIGURE 2

Photos showing Congo red staining results (A) and clear zone diameters, colony diameters, and ratios of clear zone diameters and colony diameters (B) of some strains. (A) Congo red staining of some strains. (B) Congo red stained clear zone size.

FIGURE 3

Activities of cellulase, including carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), and β-glucosidase (β-Gase), of the seven cellulose-degrading bacterial strains. Different letters indicate significant differences between strains for a same enzyme (P < 0.05).

FIGURE 4

Colony and cell morphologies of the seven isolated cellulase-producing strains. (A) Colony morphology. (B) Cell morphology.

FIGURE 5

Phylogenetic trees of the seven isolated cellulase-producing microbial strains based on 16S rDNA (bacteria) and D1/D2 domains of the 28S rDNA (fungi) sequences. (A) Bacteria. (B) Fungi.

FIGURE 6

Photos showing the results of the antagonism test between the seven cellulase-producing strains.

FIGURE 7

Optimization of culture conditions for cellulase production by microbial consortium VI by the single factor approach. In Panel (A), the temperature was 30°C, the pH was 7, and the inoculum volume was 5% (v/v); in Panel (B), the culture time was 4 days, the pH was 7, and the inoculum volume was 5% (v/v); in Panel (C), the temperature was 35°C, the culture time was 4 days, and the inoculum volume was 5% (v/v); in Panel (D), the temperature was 35°C, the culture time was 4 days, and the pH was 6.5. (A) Effect of time on enzyme production by consortium. (B) Effect of temperature on enzyme production by consortium. (C) Effect of initial pH on enzyme production by consortium. (D) Effect of inoculation volume on enzyme production by consortium. Error bars represent standard deviation; they were calculated using data from 3 technical replicates. Different letters (e.g., “a,” “b,” “c,” and “d”) represented significant difference (p < 0.05).

FIGURE 8

Response surface plots showing the interaction effects between factors on carboxymethyl cellulase (CMCase) activity of consortium VI. (A) The effect of the interaction between culture time and culture temperature on enzyme activity. (B) The effect of the interaction between culture time and pH on enzyme activity. (C) The effect of the interaction between culture time and inoculum volume on enzyme activity. (D) The effect of the interaction between culture temperature and pH on enzyme activity. (E) The effect of the interaction of culture temperature and inoculum volume on enzyme activity. (F) The effect of the interaction of pH and inoculum volume on enzyme activity. Different letters (e.g., “a,” “b,” “ab,” and “c”) in a same column indicate significant differences at p < 5%.