The microbial community responsible for dechlorination and benzene ring opening during anaerobic degradation of 2,4,6‑trichlorophenol

Highlights

• 2,4,6-TCP-degrading biomass can be obtained by controlling culture conditions.

• HiSeq sequencing analyses and special functional assays were conducted.

• Abundance of H₂ production-associated bacteria explains rapid TCP dechlorinating.

• Ignavibacterium was presumed to be responsible for benzene ring opening.

Abstract

This study describes the dechlorination ability of acclimated biomass, the high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene of such microorganisms, and the analysis of their community structure in relation to special functions. Two types of acclimated biomass (AB-1 and AB-2) were obtained via different acclimated treatment processes and were used to degrade 2,4,6‑trichlorophenol. The degradation pathway and characteristics of trichlorophenol degradation were different between the two groups. AB-1 degraded trichlorophenol only to 4-chlorophenol. AB-2 completely dechlorinated trichlorophenol and opened the benzene ring. The 16S rRNA high-throughput sequencing method was employed to examine the microbial diversity. It was found that the microbial richness and diversity of AB-1 were higher than those of AB-2. Firmicutes and Bacteroidetes were 2.7-fold and 4.3-fold more abundant, respectively, in AB-1 than in AB-2. Dechlorination bacteria in AB-1 mainly included Desulfobulbus, Desulfovibrio, Dechloromonas, and Geobacter. The above-mentioned bacteria were less abundant in AB-2, but the abundance of Desulfomicrobium was twofold higher in AB-2 than in AB-1. The two types of acclimated biomass contained different hydrogen (H₂)-producing bacteria. AB-2 showed higher abundance and diversity of hydrogen-producing bacteria. There was no Ignavibacteriae in AB-1, whereas its abundance in AB-2 was 8.4%. In this biomass, Ignavibacteriae was responsible for opening of the benzene ring. This study indicates that the abundance and diversity of microorganisms are not necessarily beneficial to the formation of a functional dechlorinating community. The H₂-producing bacteria (which showed greater abundance and diversity) and Ignavibacterium were assumed to be core functional populations that gave AB-2 stronger dechlorination and phenol-degradation abilities. Control of lower oxidation reduction potential (E_h) and higher temperatures by means of fresh aerobic activated sludge as the starting microbial group, caused rapid complete dechlorination of 2,4,6‑trichlorophenol and benzene ring opening.

Introduction

2,4,6‑Trichlorophenol (TCP) is a cause for serious environmental concern because of its widespread application as a solvent, plasticizer, and insecticide and its presence at many hazardous waste sites (Fricker et al., 2014; Limam et al., 2016). It is detected in wastewater, surface waters, groundwater, and excess sludge from wastewater treatment plants. TCP was classified as a priority pollutant by the US Environmental Protection Agency (US Department of Health and Human Services Public Health Service, 2011). TCP can also inhibit biological treatment systems owing to its biocidal effect.

The most important pathway of anaerobic biodegradation of chlorophenolic organics is anaerobic reductive dechlorination (Loffler et al., 2013; Mikesell and Boyd, 1986; J. Song et al., 2015). That is, chlorophenol (CP) serves as an electron acceptor, and the chlorine atoms on the benzene ring are removed simultaneously. The chlorine atoms in polychlorinated phenol are removed one by one to form mono-CP. Then, the last chlorine atom in mono-CP is removed, and phenol is formed. Finally, the phenol ring is opened and mineralized into CH₄ and CO₂. The number of chlorine atoms in a compound and culture conditions have a strong influence on the anaerobic biodegradability of chlorophenolic organics, thus leading to a variety of degradation pathways, intermediate products, end products, degradation kinetics, and microbial communities (Denk and Milutinovic, 2018; Ghattas et al., 2017; Sohn and Haggblom, 2016).

In general, ortho‑chlorine atoms are most easily removed, meta‑chlorine comes second, and the para‑chlorine atom is the most difficult to eliminate. The greater the number of chlorine atoms in a compound, the weaker is the dechlorination ability of a microorganism. It is more difficult to open the benzene ring than to perform dechlorination. Normally, the dechlorination function of purebred bacteria is position-specific and can remove chlorine only from a certain position (Fricker et al., 2014; Li et al., 2013b; Puyol et al., 2011; Sun et al., 2000); the examples are Dehalococcoides, Desulfovibrio dechloroacetivorans, Desulfovibrio thermophilum, and Dehalobacter strains. It is possible to achieve complete dechlorination, benzene ring opening, and subsequent mineralization by means of a mixture of microorganisms. Acclimated activated sludge usually can perform more thorough degradation of polychlorinated organic compounds. For chlorinated organic compounds containing benzene ring structures, the final degradation products are often some low-chlorinated organic compounds or phenol. For example, polychlorinated biphenyls are generally degraded to monochlorobenzene and dichlorobenzene (Zhen et al., 2014). The degradation products of polychlorinated phenols often include a variety of low-chlorinated phenols (Limam et al., 2016; Yoshida et al., 2007). TCP tends to be degraded to p-chlorophenol (p-CP) or phenol (Li et al., 2013a; Mun et al., 2008; Puyol et al., 2011; J. Song et al., 2015). p-CP is a common product because para‑chlorine in p-CP is difficult to remove by a nucleophilic attack. Therefore, p-CP is often not easily degraded and remains among the products. Nevertheless, anaerobic dechlorination usually is not complete because the mono-CPs are resistant to reductive dechlorination, and benzene ring opening is difficult to achieve at the same time.

Obviously, for a mixture of microorganisms, the function of the microbial community is crucial. Commonly, dechlorination bacteria can be obtained by an enrichment method from contaminated or pristine anoxic environments including sediments, soils, aquifers, and sewage sludge (Matus et al., 1996; Smidt and de Vos, 2004). Nevertheless, until 2007 (Adrian et al., 2007; Aulenta et al., 2007) only a few strains of Desulfitobacterium has been known to dechlorinate highly chlorinated phenols. Lately, a variety of dechlorinating bacteria (Fricker et al., 2014; Johnson et al., 2009; Jung et al., 2013; Loffler et al., 2013; Lu et al., 2010; Zhen et al., 2014) were isolated and identified. Among them, some strains of Dehalococcoides, Dehalobacter, Clostridium, and Desulfitobacterium can perform the dechlorination function well. Dehalococcoides represents excellent dechlorinators, which are obligate organohalide-respiring bacteria. These dechlorinating microorganisms mainly dechlorinate substances such as trichloroethylene and perchlorate.

In contrast, for harmless disposal of halogenated aromatic hydrocarbons, chlorine atom removal and benzene ring opening are necessary. Sohn and colleagues (Sohn and Haggblom, 2016; Sohn et al., 2018; Zhen et al., 2014) investigated the degradation of polychlorinated biphenyls and the relevant microbial community by PCR with denaturing gradient gel electrophoresis. In that work, benzene ring opening was not achieved. The microbial community included the aforementioned dechlorinating microorganisms; their studies have not uncovered the microorganisms capable of benzene ring opening. One research group (Wang et al., 2017) studied reductive dechlorination of hexachlorobenzene. The results showed that hexachlorobenzene is not eventually opened. The formation of a specific functional microbial population should be related to many factors such as inoculated sludge, culture temperature, and other relevant parameters. Some studies suggest that inoculation of sludge and culture conditions are crucial for the formation of functional communities (Song et al., 2011; Tao et al., 2013). Another research group (Zhou et al., 2006) found that a higher culture temperature (38 °C) and low oxidation reduction potential (E_h < −200 mV) increase the rate of dechlorination of penta-CP. These conditions induce the differences between microbial communities and then yield different functions. Some investigators (Yoshida et al., 2007) analyzed several microbial communities that degrade halogenated organic compounds containing benzene rings. Nevertheless, there are few studies using Illumina MiSeq to analyze such microbial communities. Thus, complete dechlorination and ring opening are important for the halogenated-organic-matter degradation. Although some researchers have a good understanding of anaerobic dechlorination microorganisms, the studies on the population and diversity of TCP-degrading microbes are still lacking. Therefore, before moving on to further studies involving CPs as a contaminant, it was necessary to determine why the dechlorination pathways are different and understand how to identify and control the core microbial community. Hence, a comparative analysis has great scientific significance.

In this study, we report that dechlorinating biomass can be obtained from sludge with a high rate of degradation and a complete degradation pathway via control of culture conditions. The acclimated treatment process determined the dechlorination rate and degradation extent. We performed Miseq sequencing, analyzed the microbial community structure, and revealed that the different functional microbial groups engaged in multiple syntrophic relationships in the biomass TCP-degrading reactors.