Stable-Isotope Probing-Enabled Cultivation of the Indigenous Bacterium Ralstonia sp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater

Abstract

To identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (Vogesella and Alicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereas Bacillus and Cupriavidus were involved in BP degradation. Vogesella and Alicyclobacillus have not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity of Ralstonia-like degraders involved in both PHE and BP degradation. To target the separation of functional Ralstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities. IMPORTANCE The comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.

Decoupled Spatial Distribution of PAHs Degraders Determined by Taxonomic 16S rRNA and Degrading Genes Across Chinese Forest Soils

“…Other soil properties including pH, electric conductivity (EC), ammonium nitrogen (NH 4 ‐N), total phosphorus (TP), available phosphorus (AP), total potassium (TK), available potassium (AK), aluminum (Al), calcium (Ca), and magnesium (Mg) were measured following the standard methods in Bao Shidan (2000). PAHs were analyzed using the methods described previously (Li et al, 2019; Song et al, 2016) with some modifications. Briefly, 20 g of soil sample was subjected to freeze drying, pulverizing, and extraction with dichloromethane (DCM).…”

“…SIP microcosms were prepared as previously described with some modifications (Li et al, 2019; Song et al, 2015, 2016). Briefly, 3.0 g of soil (dry weight) was placed in a 150‐ml serum bottle containing 10 ml of phosphate‐buffered mineral medium.…”

“…The DNA content was quantified with an ND‐2,000 UV‐Vis spectrophotometer (NanoDrop Technologies, USA). Thereafter, equilibrium density gradient centrifugation was performed to separate the 13 C‐labeled DNA from the unlabeled DNA according to a protocol described by Li and Song (Li et al, 2019; Song et al, 2015). Following centrifugation, the centrifuge tubes were placed on a fraction recovery system (Beckman Coulter, USA), and fractions were collected.…”

“…The copy numbers of PAH‐RHD α genes in fractions from the 12 C‐PT and 13 C‐PT microcosms were determined by quantitative polymerase chain reaction (qPCR) using a primer set of 396F (5′‐ATTGCGCTTAYCAYGGBTGG‐3′) and 696R (5′‐ATAGGTGTCTCCAACRAARTT‐3′) as described previously (Ding et al, 2010). The qPCR and the construction of the standard curve were carried out according to the protocol described previously (Li et al, 2019). The “light” and “heavy” DNA fractions were selected based on the copy numbers of PAH‐RHD α genes in the 12 C‐PT and 13 C‐PT microcosms and then subjected to the following Illumina sequencing.…”

“…The 16S rRNA gene sequencing data were processed and analyzed using the Mothur (v.1.34.4) (Schloss et al, 2009) and Quantitative Insights Into Microbial Ecology (QIIME 1) (Caporaso et al, 2010) following the published procedure (Edgar, 2010; Jiang et al, 2016; Li et al, 2019; McDonald et al, 2012). Mothur was employed to assemble the raw sequences and search the chimera in the aligned sequences.…”