Biodegradation Capacity and Activity Enzymatic of Bacillus subtilis against Low-Density Polyethylene

Aims: The objective of this work was to determine the degradation capacity of low-density polyethylene by the bacterium Bacillus subtilis and analyze the production of extracellular laccase activity. Methodology: The experiments was realized in 50 mL of culture medium, added with a fragment of known dry weight (1 cm2 colorless polyethylene bag squares), and were incubated at 28°C, pH 6.5, for 6 months under static conditions, determining the growth of the bacterium by dry weight (68, 75, and 91 mg), the production of extracellular protein (271, 234, and 326.1 mg/mL), and the degradation of the substrate by dry biodegraded (8.57%, 5.88%, and 11.76%). Results: The production of extracellular laccase enzyme was analyzed in presence of polyethylene, finding an enzymatic activity of laccase of 2.06, 1.49, and 2,03 U/mL, while in the control without substrate, no enzymatic activity was observed, which suggests that this enzyme may participate in the degradation of polyethylene. In addition, some characteristics of the extracellular enzymatic activities were analyzed, such as stability at 4oC and 28oC, optimal pH and temperature, the effect of protein and substrate concentration. Conclusion: The extracellular protein production and dry weight of the bacterium are higher in the presence of low-density polyethylene. The laccase activity is very stable at 4oC and 28oC, the most effective pH and temperature, were 4.5 and 28oC, and present an incubation time of 5 minutes, and this data suggest that this enzymatic activitiy may participate in the degradation of low density polyethylene.

Bisphenol A Removal by the Fungus Myrothecium roridumIM 6482—Analysis of the Cellular and Subcellular Level

Bisphenol (BPA) is a key ingredient in the production of epoxy resins and some types of plastics, which can be released into the environment and alter the endocrine systems of wildlife and humans. In this study, the ability of the fungus M. roridumIM 6482 to BPA elimination was investigated. LC-MS/MS analysis showed almost complete removal of BPA from the growth medium within 72 h of culturing. Products of BPA biotransformation were identified, and their estrogenic activity was found to be lower than that of the parent compound. Extracellular laccase activity was identified as the main mechanism of BPA elimination. It was observed that BPA induced oxidative stress in fungal cells manifested as the enhancement in ROS production, membranes permeability and lipids peroxidation. These oxidative stress markers were reduced after BPA biodegradation (72 h of culturing). Intracellular proteome analyses performed using 2-D electrophoresis and MALDI-TOF/TOF technique allowed identifying 69 proteins in a sample obtained from the BPA containing culture. There were mainly structural and regulator proteins but also oxidoreductive and antioxidative agents, such as superoxide dismutase and catalase. The obtained results broaden the knowledge on BPA elimination by microscopic fungi and may contribute to the development of BPA biodegradation methods.

Effect of Pretreated Colza Straw on the Growth and Extracellular Ligninolytic Enzymes Production by Lentinula edodes and Ganoderma lucidum

Lentinula edodes 3565 and Ganoderma lucidum 9621 were compared for their ability to produce lignocellulolytic enzymes in submerged (SM) and surface liquid (SL) fermentation of hydrolysed colza straw lignin waste that remained after the production of furfural and bioethanol (CS lignin). Application of cultivated mushrooms to dispose of pretreated colza straw agricultural waste is an approach to decrease the quantity of residual lignin while simultaneously obtaining active substances, e.g., the ligninolytic enzyme complex from mycelium. The effect of adding CS lignin to culture media on the yield of L. edodes and G. lucidum mycelium and extracellular laccase activity was studied. It was revealed that the mycelial growth of G. lucidum on solid media was significantly improved by adding CS lignin. Laccase activity during SL cultivation of L. edodes on medium with CS lignin gradually increased over the experiment starting on day 21 and peaked at 520 U/mL on day 28. G. lucidum expressed the maximum laccase activity, 540 U/mL, during the first 14 days of mycelium SM cultivation. Extracellular laccase activity was enhanced about 35- to 40-fold at cultivation of L. edodes and about 10- to 15-fold in the case of G. lucidum by supplementing liquid culture media with CS lignin.

Comparative Study of Secreted Proteins, Enzymatic Activities of Wood Degradation and Stilbene Metabolization in Grapevine Botryosphaeria Dieback Fungi

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccum parvum and Diplodia seriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N.parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases.

Extra and intracellular laccase titers of xylophagic bacteria isolated from adult termite

Laccase is an important enzyme in terms of its versatile applicability, but its commercial use is limited by factors such as high production cost, low activity and/or stability under given conditions. The objective of this study was to screen xylophagic bacteria isolated from termites for the production of extracellular and intracellular laccases. Six laccase-positive strains were isolated, namely CA, A3, A5, A6, A7 and A8. They were molecularly identified by sequence analysis of 16S rRNA and classified under the genera Bacillus (A7, A8, CA) and Pseudomonas (A3, A5, A6). Laccase was produced by these bacterial isolates by submerged fermentation and was optimized at 37°C, pH 5.5, 6.2 and 7.0, with agitation and 0.5 mM guaiacol (as carbon source). Laccase activity was determined by measuring the oxidation of guaiacol and ABTS (2,21-azino bis[3-ethylbenzthiazoline-6-sulfonate]). Strain A5 produced extracellular laccase titers ranging from 123 to 168 U ml-1. Guaiacol was identified as a better substrate for the quantification of laccase. In conclusion, bacteria harboring the gut of termites can produce extracellular laccase with activity at medium to moderate acidity.

Efficient extracellular laccase secretion via bio-designed secretory apparatuses to enhance bacterial utilization of recalcitrant lignin

Our study advances the knowledge of secretion mechanisms in Gram-negative bacteria and provides novel insights into the lignin utilization by extracellular lignolytic enzyme-bacterial cell systems.