William M. Moe

Dehalogenimonas lykanthroporepellens gen. nov., sp. nov., a reductively dehalogenating bacterium isolated from chlorinated solvent-contaminated groundwater.

Two recently reported bacterial strains that are able to reductively dehalogenate polychlorinated aliphatic alkanes, including 1,2,3-trichloropropane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane, were further characterized to clarify their taxonomic position. The two strains, designated BL-DC-8 and BL-DC-9(T), were mesophilic, non-spore-forming, non-motile, Gram-negative staining and strictly anaerobic. Cells were irregular cocci, 0.3-0.6 mum in diameter. The two strains were resistant to ampicillin and vancomycin. Hydrogen was utilized as an electron donor. The genomic DNA G+C content of strains BL-DC-8 and BL-DC-9(T) was 54.0 and 53.8 mol%, respectively. The major cellular fatty acids were C(18 : 1)omega9c, C(16 : 1)omega9c, C(16 : 0) and C(14 : 0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that the strains cluster within the phylum Chloroflexi, but are related only distantly to all recognized taxa in the phylum. Morphological, physiological and chemotaxonomic traits as well as phylogenetic analysis support the conclusion that these two strains represent a novel species of a new genus in the phylum Chloroflexi, for which the name Dehalogenimonas lykanthroporepellens gen. nov., sp. nov. is proposed. The type strain of Dehalogenimonas lykanthroporepellens is BL-DC-9(T) (=ATCC BAA-1523(T) =JCM 15061(T)).

Biodegradation of volatile organic compounds by five fungal species.

Abstract. Five fungal species, Cladosporium resinae (ATCC 34066), Cladosporium sphaerospermum (ATCC 200384), Exophiala lecanii-corni (CBS 102400), Mucor rouxii (ATCC 44260), and Phanerochaete chrysosporium (ATCC 24725), were tested for their ability to degrade nine compounds commonly found in industrial off-gas emissions. Fungal cultures inoculated on ceramic support media were provided with volatile organic compounds (VOCs) via the vapor phase as their sole carbon and energy sources. Compounds tested included aromatic hydrocarbons (benzene, ethylbenzene, toluene, and styrene), ketones (methyl ethyl ketone, methyl isobutyl ketone, and methyl propyl ketone), and organic acids (n-butyl acetate, ethyl 3-ethoxypropionate). Experiments were conducted using three pH values ranging from 3.5 to 6.5. Fungal ability to degrade each VOC was determined by observing the presence or absence of visible growth on the ceramic support medium during a 30-day test period. Results indicate that E. lecanii-corni and C. sphaerospermum can readily utilize each of the nine VOCs as a sole carbon and energy source. P. chrysosporium was able to degrade all VOCs tested except for styrene under the conditions imposed. C. resinae was able to degrade both organic acids, all of the ketones, and some of the aromatic compounds (ethylbenzene and toluene); however, it was not able to grow utilizing benzene or styrene under the conditions tested. With the VOCs tested, M. rouxii produced visible growth only when supplied with n-butyl acetate or ethyl 3-ethoxypropionate. Maximum growth for most fungi was observed at a pH of approximately 5.0. The experimental protocol utilized in these studies is a useful tool for assessing the ability of different fungal species to degrade gas-phase VOCs under conditions expected in a biofilter application.

Effect of nitrogen limitation on performance of toluene degrading biofilters.

The literature reports conflicting observations regarding the need for nutrient addition to biofilters treating contaminated gases. Such conflicts are often based on quasi-steady-state performance data collected on biofilters operated under continuous loading conditions. In the studies described herein, the impact of nitrogen limitations on two toluene-fed biofilters was assessed over a 97-day period. The biofilters were packed with polyurethane foam medium and contained different initial levels of nitrate-nitrogen. Toluene and CO2 concentration profiles were monitored during both normal steady loading conditions and short-term, unsteady-state transient loading conditions (e.g., shock loads). Packing medium samples were periodically removed and analyzed to quantify changes in nitrate-nitrogen content over time. Data are presented which show that over long-time periods (several months), nutrient-induced kinetic limitations diminished biofilter performance during transient, unsteady-state conditions even when performance during normal steady loading was not adversely affected. Elemental analysis of biomass removed from the biofilters support nitrate-nitrogen and CO2 concentration profile data and clearly illustrate how kinetically limited biofilters fail during shock loads even when there is an overall stoichiometric excess of nutrients.

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9T) and comparison to “Dehalococcoides” strains

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9T contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus.

Dehalogenimonas alkenigignens sp. nov., a chlorinated-alkane-dehalogenating bacterium isolated from groundwater.

Two strictly anaerobic bacterial strains, designated IP3-3(T) and SBP-1, were isolated from groundwater contaminated by chlorinated alkanes and alkenes at a Superfund Site located near Baton Rouge, Louisiana (USA). Both strains reductively dehalogenate a variety of polychlorinated aliphatic alkanes, including 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane and 1,2,3-trichloropropane, when provided with hydrogen as the electron donor. To clarify their taxonomic position, strains IP3-3(T) and SBP-1 were characterized using a polyphasic approach. Both IP3-3(T) and SBP-1 are mesophilic, non-spore-forming, non-motile and Gram-stain-negative. Cells of both strains are irregular cocci with diameters of 0.4-1.1 µm. Both are resistant to ampicillin and vancomycin. The genomic DNA G+C contents of strains IP3-3(T) and SBP-1 are 55.5±0.4 and 56.2±0.2 mol% (HPLC), respectively. Major cellular fatty acids include C18 : 1ω9c, C16 : 0, C14 : 0 and C16 : 1ω9c. 16S rRNA gene sequence based phylogenetic analyses indicated that the strains cluster within the phylum Chloroflexi most closely related to but distinct from the species Dehalogenimonas lykanthroporepellens (96.2 % pairwise similarity) and Dehalococcoides mccartyi (90.6 % pairwise similarity). Physiological and chemotaxonomic traits as well as phylogenetic analysis support the conclusion that these strains represent a novel species within the genus Dehalogenimonas for which the name Dehalogenimonas alkenigignens sp. nov. is proposed. The type strain is IP3-3(T) ( = JCM 17062(T) = NRRL B-59545(T)).

Detection and Quantification of Dehalogenimonas and “Dehalococcoides” Populations via PCR-Based Protocols Targeting 16S rRNA Genes

ABSTRACT Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to “Dehalococcoides” but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.

Dehalogenimonas spp. can Reductively Dehalogenate High Concentrations of 1,2-Dichloroethane, 1,2-Dichloropropane, and 1,1,2-Trichloroethane.

The contaminant concentrations over which type strains of the species Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens were able to reductively dechlorinate 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were evaluated. Although initially isolated from an environment with much lower halogenated solvent concentrations, D. alkenigignens IP3-3T was found to reductively dehalogenate chlorinated alkanes at concentrations comparable to D. lykanthroporepellens BL-DC-9T. Both species dechlorinated 1,2-DCA, 1,2-DCP, and 1,1,2-TCA present at initial concentrations at least as high as 8.7, 4.0, and 3.5 mM, respectively. The ability of Dehalogenimonas spp. to carry out anaerobic reductive dechlorination even in the presence of high concentrations of chlorinated aliphatic alkanes has important implications for remediation of contaminated soil and groundwater.

Biofilter Treatment of Volatile Organic Compound Emissions from Reformulated Paint: Complex Mixtures, Intermittent Operation, and Startup

Abstract Two biofilters were operated to treat a waste gas stream intended to simulate off-gases generated during the manufacture of reformulated paint. The model waste gas stream consisted of a five-component solvent mixture containing acetone (450 ppmv), methyl ethyl ketone (12 ppmv), toluene (29 ppmv), ethylbenzene (10 ppmv), and p-xylene (10 ppmv). The two biofilters, identical in construction and packed with a polyurethane foam support medium, were inoculated with an enrichment culture derived from compost and then subjected to different loading conditions during the startup phase of operation. One biofilter was subjected to intermittent loading conditions with contaminants supplied only 8 hr/day to simulate loading conditions expected at facilities where manufacturing operations are discontinuous. The other biofilter was subjected to continuous contaminant loading during the initial start period, and then was switched to intermittent loading conditions. Experimental results demonstrate that both startup strategies can ultimately achieve high contaminant removal efficiency (>99%) at a target contaminant mass loading rate of 80.3 g m−3 hr−1 and an empty bed residence time of 59 sec. The biofilter subjected to intermittent loading conditions at startup, however, took considerably longer to reach high performance. In both biofilters, ketone components (acetone and methyl ethyl ketone) were more rapidly degraded than aromatic hydrocarbons (toluene, ethylbenzene, and p-xylene). Scanning electron microscopy and plate count data revealed that fungi, as well as bacteria, populated the biofilters.

Production of hydrogen by Clostridium species in the presence of chlorinated solvents

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.

Pelosinus defluvii sp. nov., isolated from chlorinated solvent-contaminated groundwater, emended description of the genus Pelosinus and transfer of Sporotalea propionica to Pelosinus propionicus comb. nov.

Two anaerobic bacterial strains, designated SHI-1(T) and SHI-2, were isolated from chlorinated solvent-contaminated groundwater. They were found to be identical in phenotypic properties and shared high (98.5-99.8 %) pairwise 16S rRNA gene sequence similarity. Multiple 16S rRNA genes were found to be present in the isolates as well as Pelosinus fermentans DSM 17108(T) and Sporotalea propionica DSM 13327(T). Strains SHI-1(T) and SHI-2 could be differentiated from their closest phylogenetic relatives, P. fermentans DSM 17108(T) and S. propionica DSM 13327(T), on the basis of their phenotypic and phylogenetic properties. The isolates were Gram-negative, spore-forming, motile rods with peritrichous flagella. Growth occurred at 10-42 °C and pH 5.5-8.5. Fermentative growth was observed on Casamino acids, fructose, fumarate, glucose, glycerol, pyruvate and yeast extract. The major organic acids produced from glucose and glycerol fermentation were propionate and acetate. The major organic acids produced from fermentation of fumarate were propionate, acetate and succinate. The major cellular fatty acids were summed feature 4 (consisting of C(15:1)ω8c and/or C(15:2)), summed feature 8 (consisting of C(17:1)ω8c and/or C(17:2)) and C(14:0) dimethyl aldehyde. The polar lipids comprised aminophospholipids, including phosphatidylethanolamine and phosphatidylserine, and an unknown phospholipid. The genomic DNA G+C content was 39.2 mol%. We propose that strains SHI-1(T) and SHI-2 are assigned to a novel species of the genus Pelosinus, with the name Pelosinus defluvii sp. nov. (type strain SHI-1(T) = NRRL Y-59407(T) = LMG 25549(T)). The description of the genus Pelosinus is emended. We also propose the transfer of S. propionica to the genus Pelosinus as Pelosinus propionicus comb. nov. (type strain TmPN3(T) = DSM 13327(T) = ATCC BAA-626(T)), on the basis of phylogenetic, chemotaxonomic and phenotypic properties.