Isolation and molecular identification of lignin peroxidase-producing bacterial isolates from Jeddah City
Authors: Batayyib Reem S.M. , Al-Twaty Nada H. , El-Hamshary Ola I.M. 
Published: August 2022

Background
The identification of naturally occurring bacteria with lignin-oxidizing enzymes would be significant. Several species of filamentous bacteria belonging to the genus Streptomyces (Actinomycetes) have been identified as degraders of lignin. Such species play the most important role in biodegradation of lignin.

Objective
This study aimed to isolate and discover promising isolates and ideal conditions for lignin peroxidase (LiP) production as well as 16S-rRNA identification of the ligninolytic bacterial strains.

Materials and methods
Lignin was isolated and purified from black wood liquor. The ligninolytic bacterial colonies were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA. Fermentation medium (FM) was used for screening of lignin-degrading bacteria after dilution of the soil sample using lignin (0.1% v/v). The FM medium was supplemented with 50 mg/l of Azure B and toluidine dyes and 100 mg/l of tannic acid. FM was used without any supplements and agar for isolation of lignin-degrading bacteria after dilution of the soil samples. Different concentrations of lignin (0.1–0.9%) were applied to optimize LiP production by the selected strains under different temperatures (30, 35, 40, and 45°C); different pH values (7, 7.5, 8.0, and 8.5); eight different carbon sources (0.1%, w/v), such as glucose, fructose, xylose, lactose, sucrose, carboxymethyl cellulose, and xylan; and four organic sources (0.1%, w/v), such as peptone, meat extract, sodium nitrate, and potassium nitrate. The enzyme productivity was evaluated in the culture supernatant. The bacterial strain genomic DNA was extracted from pure culture isolated from soil and subjected to amplification and sequencing of 16 S ribosomal RNA gene.

Results and discussion
Nine ligninolytic bacterial colonies that excrete peroxidases based on the use of lignin (as sole carbon source) were isolated from three types of soil farms (F1, F2, and F3) from Jeddah, KSA, and the promising isolates and the optimum conditions for LiP production using FM under three incubation periods were evaluated. Two most active isolates for production of LiP belonging to Actinomycetes and Bacilli designated (R-St-1 and R-B-1) were identified using 16S-rRNA. Results showed that the highest LiP producer was
Streptomyces R-St-1
isolate (3.8 U/ml) followed by Bacilli R-B-1 isolate (2.4 U/ml) after 3 days of fermentation. Different concentrations of lignin (0.1–0.9%) were tested for their effect on LiP production by
Streptomyces R-St-1
. As lignin concentration increased, LiP production increased, and the maximum productivity of 4.9 U.mL−1 was observed at 0.5% lignin after which the LiP production was decreased. At the ideal temperature recorded of 35°C and at the optimum pH of 7.5, the production of LiP rose significantly (4.6 U.mL-1 and 4.0 U.mL-1). Various carbon sources were examined for LiP production, and glucose was shown to be the best option for producing a high yield of LiP by
Streptomyces R-St-1
, followed by lactose (4.6 and 4.0 U/ml, respectively). However, neither organic nor inorganic nitrogen sources were shown to be suited for high LiP output. As lignin concentration increased, LiP production increased, and the maximum productivity of 4.4 U/ml from Bacilli R-B-1 isolate was observed at 0.7% lignin, after which LiP production was decreased. The optimum temperature was 40°C, where LiP production showed a significant increase (4.5 U/ml), whereas the optimum pH was 8.0, and sucrose was found to be more suitable for high yield of LiP production followed by glucose (4.3 and 3.3 U/ml, respectively). The identified bacterial DNA sequences were conserved in the GenBank under two accession numbers OL697233.1 (Streptomyces
lavendulae R-St-1) and Priestia aryabhattai
R-B-1 (OL697234.1) (formerly known as Bacillus aryabhattai).