Performance and microbial community composition dynamics of aerobic granular sludge from sequencing batch bubble column reactors operated at 20°C, 30°C, and 35°C

Two bubble column sequencing batch reactors fed with an artificial wastewater were operated at 20°C, 30°C, and 35°C. In a first stage, stable granules were obtained at 20°C, whereas fluffy structures were observed at 30°C. Molecular analysis revealed high abundance of the operational taxonomic unit 208 (OTU 208) affiliating with filamentous bacteria Leptothrix spp. at 30°C, an OTU much less abundant at 20°C. The granular sludge obtained at 20°C was used for the second stage during which one reactor was maintained at 20°C and the second operated at 30°C and 35°C after prior gradual increase of temperature. Aerobic granular sludge with similar physical properties developed in both reactors but it had different nutrient elimination performances and microbial communities. At 20°C, acetate was consumed during anaerobic feeding, and biological phosphorous removal was observed when Rhodocyclaceae-affiliating OTU 214 was present. At 30°C and 35°C, acetate was mainly consumed during aeration and phosphorous removal was insignificant. OTU 214 was almost absent but the Gammaproteobacteria-affiliating OTU 239 was more abundant than at 20°C. Aerobic granular sludge at all temperatures contained abundantly the OTUs 224 and 289 affiliating with Sphingomonadaceae indicating that this bacterial family played an important role in maintaining stable granular structure

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides”-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes” strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidate

Reductive dechlorination of tetrachloroethene by a stepwise catalysis of different organohalide respiring bacteria and reductive dehalogenases

The enrichment culture SL2 dechlorinating tetrachloroethene (PCE) to ethene with strong trichloroethene (TCE) accumulation prior to cis-1,2-dichloroethene (cis-DCE) formation was analyzed for the presence of organohalide respiring bacteria and reductive dehalogenase genes (rdhA). Sulfurospirillum-affiliated bacteria were identified to be involved in PCE dechlorination to cis-DCE whereas "Dehalococcoides"-affiliated bacteria mainly dechlorinated cis-DCE to ethene. Two rdhA genes highly similar to tetrachloroethene reductive dehalogenase genes (pceA) of S. multivorans and S. halorespirans were present as well as an rdhA gene very similar to the trichloroethene reductive dehalogenase gene (tceA) of "Dehalococcoides ethenogenes" strain 195. A single strand conformation polymorphism (SSCP) method was developed allowing the simultaneous detection of the three rdhA genes and the estimation of their abundance. SSCP analysis of different SL2 cultures showed that one pceA gene was expressed during PCE dechlorination whereas the second was expressed during TCE dechlorination. The tceA gene was involved in cis-DCE dechlorination to ethene. Analysis of the internal transcribed spacer region between the 16S and 23S rRNA genes revealed two distinct sequences originating from Sulfurospirillum suggesting that two Sulfurospirillum populations were present in SL2. Whether each Sulfurospirillum population was catalyzing a different dechlorination step could however not be elucidated

An unusual tandem-domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell

Use of silicate minerals for long-term pH control during reductive dechlorination of high tetrachloroethene concentrations in continuous flow-through columns

The long-term buffering potential of three silicate minerals (diopside, fayalite and forsterite) present as fine particles in porous quartz sand medium was evaluated in flow-through column experiments over a period of 6.5 months. The columns were operated with PCE concentrations close to saturation and inoculated with the organohalide-respiring consortium SDC-9(TM), which is able to completely dechlorinate PCE to ethene at high concentrations. In the absence of pH buffering agents, fermentation and organohalide respiration drove the pH close to 6.1, leading to severe inhibition of PCE dechlorination. Forsterite and fayalite were able to maintain the pH close to 7.5 and 6.5, respectively, and to sustain the production of VC and ethene. Diopside gradually lost its buffering capacity during the first 84 days due to the formation of a low reactive leached layer but dechlorination to cis-DCE was still achieved. Among the three minerals tested, forsterite was identified as the best buffering agent. Its presence led to the best PCE removal performance and the highest relative abundance of Dehalococcoides. This study showed that forsterite and fayalite are promising sources of long-term pH buffering for in situ bioremediation of source-zone PCE. (c) 2014 Elsevier B.V. All rights reserved

Regulation of the Corrinoid Metabolism in Desulfitobacteria

The corrinoid cofactor is essential to organohalide respiration (OHR) as key element in virtually all reductive dehalogenases. Desulfitobacteria, and more particularly D. hafniense, represent a group of versatile OHR bacteria able to produce corrinoids de novo, as initially discovered in the genome of D. hafniense Y51 (1). Since then several desulfitobacteria genomes have been sequenced revealing that the corrinoid metabolism (biosynthesis, transport, regulation) represents a conserved trait in a highly variable genetic background in the members of this genus. A thorough analysis of corrinoid-related genes in the genomes of D. hafniense strains Y51, DCB-2 (2) and TCE1 (3) revealed the presence of eighteen distinct cobalamin riboswitches (Cbl-RS). Cbl-RS structures are known to regulate the transcription or translation of downstream located genes by corrinoid-dependent conformational changes occurring in the 5’-untranslated region of the corresponding RNA transcripts. In vitro in-line probing analysis of three significantly different riboswitches of D. hafniense TCE1 highlighted their role as regulatory elements in the corrinoid metabolism. In vivo analysis of the transcription of the directly downstream genes upon addition of various corrinoids clearly confirmed it (4). Furthermore, we successfully cultivated D. hafniense TCE1 in the complete absence of corrinoid in the medium, and preliminary HPLC analysis suggested that strain TCE1 produces several types of corrinoids. The corrinoid metabolism of desulfitobacteria have made them excellent candidates for acquiring OHR metabolism (5), while their wide distribution in OHR ecological niches suggests that this genus might be an important source of corrinoids for members of the genera Dehalococcoides and Dehalobacter. (1) Nonaka et al. (2006), J Bacteriol 188:2262. (2) Kim et al. (2012), BMC Microbiol 8:12. (3) JGI Project ID 403245, coordinated by H. Smidt (Wageningen University). (4) Choudhary et al. (2013), J Bacteriol 195:5186. (5) Duret et al. (2012), Appl Environ Microbiol 78:6121