Anaerobic biodegradation of phenanthrene and pyrene by sulfate-reducing cultures enriched from contaminated freshwater lake sediments

Dhar, Kartik; Abinandan, Sudharsanam; Sana, Tanmoy; Venkateswarlu, Kadiyala; Megharaj, Mallavarapu

doi:10.1016/j.envres.2023.116616

Cited by 5 publications

(3 citation statements)

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“…Expectedly, the naphthaleneand pyrene-degrading SRB enrichment cultures obtained from MGP soil were dominated by spore-forming, Gram-positive species of Desulfotomaculum (Dhar et al, 2022). In contrast, phenanthrene-and pyrene-degrading cultures from sediment samples were dominated by species of Desulfovibrio (Dhar et al, 2023). In both cases, the enrichments initially showed very slow growth on PAHs but showed satisfactory substrate removal and sulfate reduction activity after five serial passages.…”

Section: Understanding Resultsmentioning

confidence: 96%

“…In contrast, contaminated freshwater sediments may harbor SRB with biodegradation potential (Dhar et al, 2023;Pester et al, 2012). We have prepared a simple push-type corer with ordinary plumbing pipe to collect sediment from shallow freshwater lakes, circumventing the need for expensive instruments.…”

Section: Strategic Planningmentioning

confidence: 99%

“…Nonetheless, we lack comprehensive and coordinated protocols detailing essential steps such as inoculum preparation, enrichment establishment and maintenance, verification of viability of SRB in enrichments, validation of biodegradation by PAHs residual analysis and measurement of sulfate reduction activity. By following the protocols described here, we have successfully obtained naphthalene-, phenanthrene-, and pyrene-degrading, sulfatereducing enrichment cultures from contaminated soil or sediment samples (Dhar et al, 2022(Dhar et al, , 2023. With an anaerobic workstation or glovebox and a gas cylinder or reticulated system, SRB from soil or sediment can be enriched, maintained, and characterized in a reliable and reproducible manner.…”

Section: Introductionmentioning

confidence: 99%

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Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)–Degrading Strictly Anaerobic Sulfate‐Reducing Cultures from Contaminated Soil and Sediment

Dhar,

Venkateswarlu,

Megharaj

2024

Current Protocols

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Sulfate‐reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH‐degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH‐degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH‐degrading SRB cultures. In this set of protocols, we describe step‐by‐step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate‐reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow‐growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography–mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium‐throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH‐degrading sulfate‐reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Obtaining PAH‐degrading strictly anaerobic sulfate‐reducing enrichment cultures from contaminated soil and sedimentSupport Protocol 1: Operation and maintenance of an anaerobic workstationSupport Protocol 2: Setup of gas purging systems for preparing anoxic solutionsSupport Protocol 3: Verification of viability in slow‐growing SRB enrichment culturesSupport Protocol 4: Extraction of genomic DNA from low‐biomass culturesBasic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC‐MSBasic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB culturesBasic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue methodAlternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method

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Section: Understanding Resultsmentioning

confidence: 96%

Section: Strategic Planningmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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