Survival of prokaryotes in a polluted waste dump during remediation by alkaline hydrolysis.

A combination of culture-dependent and culture-independent techniques was used to characterize bacterial and archaeal communities in a highly polluted waste dump and to assess the effect of remediation by alkaline hydrolysis on these communities. This waste dump (Breakwater 42), located in Denmark, contains approximately 100 different toxic compounds including large amounts of organophosphorous pesticides such as parathions. The alkaline hydrolysis (12 months at pH >12) decimated bacterial and archaeal abundances, as estimated by 16S rRNA gene-based qPCR, from 2.1 × 10(4) and 2.9 × 10(3) gene copies per gram wet soil respectively to below the detection limit of the qPCR assay. Clone libraries constructed from PCR-amplified 16S rRNA gene fragments showed a significant reduction in bacterial diversity as a result of the alkaline hydrolysis, with preferential survival of Betaproteobacteria, which increased in relative abundance from 0 to 48 %. Many of the bacterial clone sequences and the 27 isolates were related to known xenobiotic degraders. An archaeal clone library from a non-hydrolyzed sample showed the presence of three main clusters, two representing methanogens and one representing marine aerobic ammonia oxidizers. Isolation of alkalitolerant bacterial pure cultures from the hydrolyzed soil confirmed that although alkaline hydrolysis severely reduces microbial community diversity and size certain bacteria survive a prolonged alkaline hydrolysis process. Some of the isolates from the hydrolyzed soil were capable of growing at high pH (pH 10.0) in synthetic media indicating that they could become active in in situ biodegradation upon hydrolysis.

Biodegradation of para-nitrophenol by Citricoccus nitrophenolicus strain PNP1(T) at high pH.

A gram-positive bacterium Citricoccus nitrophenolicus (strain PNP1(T), DSM 23311(T), CCUG 59571(T)) isolated from a waste water treatment plant was capable of effectively degrading p-nitrophenol (pNP) as a source of carbon, nitrogen and energy for growth. Degradation of pNP required oxygen and resulted in the stoichiometric release of nitrite. Strain PNP1(T) also degraded 4-chlorophenol, phenol and salicylate. pNP was degraded at pH values between 6.8 and 10.0 and at temperatures between 15-32 °C. pNP at concentrations up to 150 mg L(-1) were degraded during growth in media at pH ≤ 10, whereas 200 mg L(-1) was completely inhibitory to growth. When incubated in an NH(4)Cl-free medium (pH 10) containing both pNP and acetate, pNP is degraded with concomitant release of nitrite which was subsequently assimilated during acetate degradation. Intact cells of strain PNP1(T) suspended in NaHCO(3)/Na(2)CO(3) buffer were able to continuously degrade 200 mg L(-1) pNP over a 40 day period at pH 10.

Description of Citricoccus nitrophenolicus sp. nov., a para-nitrophenol degrading actinobacterium isolated from a wastewater treatment plant and emended description of the genus Citricoccus Altenburger et al. 2002.

A novel actinobacterium, designated PNP1(T), was isolated from a wastewater treatment plant at a pesticide factory by selective enrichment with para-nitrophenol. The strictly aerobic strain PNP1(T) grew with para-nitrophenol as the sole carbon and energy source. Metabolism of para-nitrophenol resulted in the stoichiometric release of nitrite. When incubated with both para-nitrophenol and acetate, para-nitrophenol was degraded and utilized as growth substrate prior to acetate. When grown on acetate (in the absence of ammonium) both nitrite and nitrate served as nitrogen sources, nitrate being quantitatively reduced to nitrite which accumulated in cultures during aerobic growth. Cells were coccoid and stained Gram-positive, were non-motile and did not form endospores. Colonies of strain PNP1(T) on agar medium were bright yellow, circular and smooth. The dominant menaquinone was MK-8(H(2)) (54%) and the major cellular fatty acid was anteiso C15:0 (75%). Strain PNP1(T) grew optimally at 27°C, at pH 8-8.5, at salinities 3% (w/v) NaCl, yet exhibited a substantial halotolerance with growth occurring at salinities up to 17% (w/v) NaCl. In addition to para-nitrophenol, a range of sugars, short chain fatty acids and alcohols served as electron donors for growth. The DNA G + C mol% was 68%. The genotypic and phenotypic properties suggest that strain PNP1(T) represents a novel species of the actinobacterial genus Citricoccus for which the name Citricoccus nitrophenolicus is proposed. It is the first member of this genus that has been reported to hydrolyze and grow on para-nitrophenol. The type strain is PNP1(T) (=DSM 23311(T) = CCUG 59571(T)).

Desulfitibacter alkalitolerans gen. nov., sp. nov., an anaerobic, alkalitolerant, sulfite-reducing bacterium isolated from a district heating plant.

A novel alkalitolerant, anaerobic bacterium, designated strain sk.kt5(T), was isolated from a metal coupon retrieved from a corrosion-monitoring reactor of a Danish district heating plant (Skanderborg, Jutland). The cells of strain sk.kt5(T) were motile, rod-shaped (0.4-0.6 x 2.5-9.6 microm), stained Gram-positive and formed endospores. Strain sk.kt5(T) grew at pH 7.6-10.5 (with optimum growth at pH 8.0-9.5), at temperatures in the range 23-44 degrees C (with optimum growth at 35-37 degrees C), at NaCl concentrations in the range 0-5 % (w/v) (with optimum growth at 0-0.5 %) and required yeast extract for growth. Only a limited number of substrates were utilized as electron donors, including betaine, formate, lactate, methanol, choline and pyruvate. Elemental sulfur, sulfite, thiosulfate, nitrate and nitrite, but not sulfate or Fe(III) citrate, were used as electron acceptors. The G+C content of the DNA was 41.6 mol%. Phylogenetic analyses of the sequence data for the dsrAB genes [encoding the major subunits of dissimilatory (bi)sulfite reductase] and the 16S rRNA gene placed strain sk.kt5(T) within a novel lineage in the class Clostridia of the phylum Firmicutes. Taken together, the physiological and genotypic data suggest that strain sk.kt5(T) represents a novel species within a novel genus, for which the name Desulfitibacter alkalitolerans gen. nov., sp. nov. is proposed. The type strain of Desulfitibacter alkalitolerans is sk.kt5(T) (=JCM 12761(T)=DSM 16504(T)).

Desulfovibrio alkalitolerans sp. nov., a novel alkalitolerant, sulphate-reducing bacterium isolated from district heating water.

A novel alkalitolerant, sulphate-reducing bacterium (strain RT2T) was isolated from alkaline district heating water. Strain RT2T was a motile vibrio (0.5-0.8 microm wide and 1.4-1.9 microm long) and grew at pH 6.9-9.9 (optimum at pH 9.0-9.4) and at 16-47 degrees C (optimum at 43 degrees C). The genomic DNA G+C content was 64.7 mol%. A limited number of compounds were used as electron donors with sulphate as electron acceptor, including lactate, pyruvate, formate and hydrogen/acetate. Sulphite and thiosulphate also served as electron acceptors. Based on physiological and genotypic properties, the isolate was considered to represent a novel species of the genus Desulfovibrio, for which the name Desulfovibrio alkalitolerans sp. nov. is proposed. The type strain is RT2T (=DSM 16529T=JCM 12612T). The strain is the first alkali-tolerant member of the genus Desulfovibrio to be described.