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Background <p>Polyethylene (PE) is the most widely produced polyolefin and represents a major contributor to plastic waste, largely due to its chemical stability and resistance to biological degradation.</p> <p>PE biodegradation is typically characterized as a series of oxidative metabolic processes rather than mineralization. An urgent priority is the development of integrated experimental setups that bring together physiological, biochemical, and molecular analyses under controlled cultivation conditions to unlock the metabolic traits of PE biodegradation.</p> <p>This study aims to elucidate the molecular mechanisms of commercial low-molecular-weight PE (LDPE) attack by <i>Rhodococcus opacus</i> R7 through an integrated genome-to-function approach, including growth and extracellular laccase assays, intracellular lipid quantification, gas chromatography coupled with mass spectrometry (GC–MS) analysis of compounds associated with untreated LDPE, and transcriptional profiling of oxidative enzymes.</p> Results <p>LDPE utilization was evaluated under multiple cultivation strategies (1% single-dose vs. 0.4% fed-batch), inoculum conditions, and polymer types (untreated or UV-pretreated) over 28&#xa0;days.</p> <p>Fed-batch LDPE (0.4%) supported slightly higher viable cell numbers than fixed-dose LDPE (1%), while prolonged PE pre-adaptation did not enhance growth. Extracellular laccase activity was detected under all conditions, but was more influenced by the physiological status of the inoculum than by PE concentration or pretreatment. Lipid accumulation was early detected in case of 1% PE, while the fed-batch conditions reflected the growth phase and physiological responses. GC–MS analyses revealed that LDPE oxidation products vary compared to abiotic and control samples. Specifically, fixed-dose LDPE favored a progressive change in the pattern-profile of carboxylic acids and medium- to long-chain alkanes, while fed-batch LDPE produced a heterogeneous mixture, including alcohols and ketones, consistent with a continuous and asynchronous transformation of the polymer.</p> <p>The fed-batch condition induced a broader and earlier transcriptional activation of oxidative genes, including seven laccase-like multicopper oxidases (<i>LMCOs</i>), alkane monooxygenase (<i>alkB</i>), benzoate dioxygenase (<i>benA</i>), and cytochrome P450 hydroxylase. Long-chain <i>n</i>-alkanes, particularly tetracosane (C24), strongly induced oxidative enzymes, suggesting their role as metabolic signals during PE degradation.</p> Conclusions <p>Overall, LDPE attack by <i>R. opacus</i> R7 emerges as a dynamic process shaped by substrate accessibility and cultivation strategy, highlighting its potential as a platform for controlled plastic biodegradation.</p>

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Genome-to-function integrated exploration of polyethylene biodegradation by Rhodococcus opacus R7

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月4日

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  • Jessica Zampolli,
  • Mattia Salvadori,
  • Valentina Vincenti,
  • Marina Lasagni,
  • Patrizia Di Gennaro

摘要

Background

Polyethylene (PE) is the most widely produced polyolefin and represents a major contributor to plastic waste, largely due to its chemical stability and resistance to biological degradation.

PE biodegradation is typically characterized as a series of oxidative metabolic processes rather than mineralization. An urgent priority is the development of integrated experimental setups that bring together physiological, biochemical, and molecular analyses under controlled cultivation conditions to unlock the metabolic traits of PE biodegradation.

This study aims to elucidate the molecular mechanisms of commercial low-molecular-weight PE (LDPE) attack by Rhodococcus opacus R7 through an integrated genome-to-function approach, including growth and extracellular laccase assays, intracellular lipid quantification, gas chromatography coupled with mass spectrometry (GC–MS) analysis of compounds associated with untreated LDPE, and transcriptional profiling of oxidative enzymes.

Results

LDPE utilization was evaluated under multiple cultivation strategies (1% single-dose vs. 0.4% fed-batch), inoculum conditions, and polymer types (untreated or UV-pretreated) over 28 days.

Fed-batch LDPE (0.4%) supported slightly higher viable cell numbers than fixed-dose LDPE (1%), while prolonged PE pre-adaptation did not enhance growth. Extracellular laccase activity was detected under all conditions, but was more influenced by the physiological status of the inoculum than by PE concentration or pretreatment. Lipid accumulation was early detected in case of 1% PE, while the fed-batch conditions reflected the growth phase and physiological responses. GC–MS analyses revealed that LDPE oxidation products vary compared to abiotic and control samples. Specifically, fixed-dose LDPE favored a progressive change in the pattern-profile of carboxylic acids and medium- to long-chain alkanes, while fed-batch LDPE produced a heterogeneous mixture, including alcohols and ketones, consistent with a continuous and asynchronous transformation of the polymer.

The fed-batch condition induced a broader and earlier transcriptional activation of oxidative genes, including seven laccase-like multicopper oxidases (LMCOs), alkane monooxygenase (alkB), benzoate dioxygenase (benA), and cytochrome P450 hydroxylase. Long-chain n-alkanes, particularly tetracosane (C24), strongly induced oxidative enzymes, suggesting their role as metabolic signals during PE degradation.

Conclusions

Overall, LDPE attack by R. opacus R7 emerges as a dynamic process shaped by substrate accessibility and cultivation strategy, highlighting its potential as a platform for controlled plastic biodegradation.