Resuscitation of viable but non-culturable bacteria to enhance the cellulose-degrading capability of bacterial community in composting

Abstract

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation-promoting factors (Rpfs) due to entering a viable but non-culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose-degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl-cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.

Figure 1

SDS‐PAGE profiles of recombinant Rpf protein after purification on a nickel‐nitrilotriacetic acid (Ni‐NTA) column with different concentrations (20–200 mM, increased in steps of 20 mM) of imidazole; Lanes: 1, E (eluent); 2, M (protein markers); 3–12, imidazole concentrations of 20–200 mM.

Figure 2

Krona chart based on the OTUs showing the relative abundance and diversity of bacterial community in the waste composting sample for the V3–V4 Illumina dataset; Circle from inside to outside stand for different classification levels.

Figure 3

Taxonomic composition of bacterial community at the phylum level in the compost sample (> 0.1%).

Figure 4

Taxonomic composition of bacterial community at the genus level in the compost sample (> 0.5%).

Figure 5

Neighbour‐joining phylogenetic tree of bacterial 16S rRNA gene sequences, including ten isolates unique to enrichment culture with Rpf addition and 15 of their most similar GenBank sequences. Bootstrap values (percentages based on 1000 replications) of above 50% are shown at the branch points. Methanobacterium alcaliphilum was used as an outgroup.

Figure 6

Extracellular cellulase activity of two enzymes (FPCase and CMCase) of bacterial cultures from TG and CG, as well as the ten isolates. X: no FPCase activity was detected. Each bar represents the average of three replicates in each sample with standard deviation (SD).