Nocardioides, Sediminibacterium, Aquabacterium, Variovorax, and Pseudomonas linked to carbon uptake during aerobic vinyl chloride biodegradation

Abstract

Vinyl chloride (VC) is a frequent groundwater contaminant and a known human carcinogen. Bioremediation is a potential cleanup strategy for contaminated sites; however, little is known about the bacteria responsible for aerobic VC degradation in mixed microbial communities. In attempts to address this knowledge gap, the microorganisms able to assimilate labeled carbon ((13)C) from VC within a mixed culture capable of rapid VC degradation (120 μmol in 7 days) were identified using stable isotope probing (SIP). For this, at two time points during VC degradation (days 3 and 7), DNA was extracted from replicate cultures initially supplied with labeled or unlabeled VC. The extracted DNA was ultracentrifuged, fractioned, and the fractions of greater buoyant density (heavy fractions, 1.758 to 1.780 g mL(-1)) were subject to high-throughput sequencing. Following this, specific primers were designed for the most abundant phylotypes in the heavy fractions. Then, quantitative PCR (qPCR) was used across the buoyant density gradient to confirm label uptake by these phylotypes. From qPCR and/or sequencing data, five phylotypes were found to be dominant in the heavy fractions, including Nocardioides (∼40 %), Sediminibacterium (∼25 %), Aquabacterium (∼17 %), Variovorax (∼6 %), and Pseudomonas (∼1 %). The abundance of two functional genes (etnC and etnE) associated with VC degradation was also investigated in the SIP fractions. Peak shifts of etnC and etnE gene abundance toward heavier fractions were observed, indicating uptake of (13)C into the microorganisms harboring these genes. Analysis of the total microbial community indicated a significant dominance of Nocardioides over the other label-enriched phylotypes. Overall, the data indicate Nocardioides is primarily responsible for VC degradation in this mixed culture, with the other putative VC degraders generating a small growth benefit from VC degradation. The specific primers designed toward the putative VC degraders may be of use for investigating VC degradation potential at contaminated sites.

Aerobic vinyl chloride degradation at the microbial community level

“…A variety of bacteria can aerobically cometabolize VC using methane 104,105 or ethene 11,13,14 as the primary substrate, and even utilize VC as the sole carbon source (VC-assimilating bacteria). Known VC-assimilating bacteria include strains of Mycobacterium 10,11,71,72 , Nocardioides 9, 10 , Pseudomonas 17,106 , 64,65 . Here, we advance our understanding of VC-assimilating bacteria in mixed cultures by tracking microbial community changes in groundwater microcosms as they adapt to VC as the sole carbon and energy source with shot-gun metagenomic sequencing.…”

“…Quantified DNA extracts (∼10 μg) were processed and centrifuged as described previously 64,65 , which includes mixing with Tris-EDTA (pH 8.0)-CsCl solution, sealing of centrifuge tubes and centrifuge for about 48 hrs at 178,000 × g (20 °C). Following ultracentrifugation, the tubes were placed onto a fraction recovery system (Beckman Coulter, Indianapolis, IN, USA), and 16 fractions (a total of 150 μL) were collected for each culture.…”

“…Following ultracentrifugation, the tubes were placed onto a fraction recovery system (Beckman Coulter, Indianapolis, IN, USA), and 16 fractions (a total of 150 μL) were collected for each culture. The BD of each fraction was measured, and CsCl was removed by glycogen-assisted ethanol precipitation 64 . Fractions were re-purified using QIAquick PCR Purification Kit as necessary.…”

“…The qPCR primer sets (Table AI. used except that the primer specificity stringency was set to four total mismatches and four mismatches within the last seven base pairs at the 3′ end 77 . The specificity of these four primer sets was tested by cloning and sequencing amplicons from G1-TP3-VC (the same culture of group A day 3 in SIP experiment), using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) and the cloning procedures described previously 64 . All the sequenced clones (six to ten clones for each primer set) were the desired targeting genus based on RDP classifier 131 (Table AI. followed by an amplicon melting program from 55°C to 95°C.…”

“…CHAPTER III. IDENTIFYING BACTERIA LINKED TO CARBON UPTAKE FROM VINYL CHLORIDE IN GROUNDWATER ENRICHMENT CULTURES USING STABLE ISOTOPE PROBING (SIP)This is a collaborative work published64 with Michigan State University. This work, together with another study65 , was focused on enrichment cultures from a contaminated groundwater plume at Carver, MA.…”