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Two novel nicotine-degrading bacterial strains were isolated from tobacco waste and identified as Acinetobacter sp. TW and Sphingomonas sp. TY based on morphology, physiological and biochemical tests, Biolog analysis and 16S rDNA sequencing. The 16S rDNA sequences have been deposited in GenBank under the accession numbers FJ753401 for TW and FJ754274 for TY. The best culture conditions for nicotine degradation were 25-37°C and pH 7.0-8.0 for strain TW and 25-30°C and pH 6.0-7.0 for strain TY. Under the best conditions, the cell growth and nicotine-degradation kinetics of the two isolates were assessed, and 1.0 g/l nicotine was completely degraded within 12 and 18 h for TW and TY, respectively. Moreover, the presence of four widely-used commercial neonicotinoid insecticides in the medium had no effects on nicotine degradation by TW; among the four tested neonicotinoids, only thiamethoxam significantly delayed nicotine degradation by TY. TW and TY were also able to degrade selected neonicotinoids. This is the first report of nicotine degradation by Acinetobacter sp. and Sphingomonas sp. This study showed that these two newly isolated bacteria may be suitable for the disposal of tobacco waste and the reduction of nicotine in tobacco leaves.

Attempts were made to acquire a plasmid-loss mutant via various methods (spontaneous mutation, SDS, and mitomycin C), among which the method involving mitomycin C (10 microg/mL) has been proven successful. Concomitant with the loss of the plasmid in Pseudomonas sp. strain HF-1, the cured derivative was identified as having a nicotine-negative (Nic-) phenotype, named mutant strain 6-13 (Nic-). After plasmids were transferred from strain HF-1 (named plasmid pMH1) to the mutant strain 6-13, the mutant strain acquired nicotine-degrading ability, called 6-13 transformant (Nic+). There were no differences in growth or nicotine-degrading efficiency between strain HF-1 (wild-type strain) and strain 6-13 transformant. After pMH1 was transferred to Escherichia coli strain Top10 (Nic-), a distant relative of Pseudomonas, it also gained nicotine-degrading ability, showing the highest nicotine degradation efficiency at pH 7.0, the optimal pH for growth of E. coli. The hsp gene, which encodes 6-hydroxy-3-succinoylpyridine hydroxylase, is involved in nicotine degradation in Pseudomonas putida strain S16 and was present in pMH1 but not in pAO1, the well-known nicotine degradation plasmid in Arthrobacter nicotinovorans. It was demonstrated that plasmid pMH1 is a novel nicotine-degrading plasmid.

Oxidative stress induced by toxic pollutants is generally responsible for the poor performance of many sequencing batch reactors (SBRs) treating organic wastewater. In this study, the oxidative stress in two SBR systems that dealt with tobacco wastewater was monitored by measuring four popular biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione, GSH; and malondialdehyde, MDA). In the non-BA (non-bio-augmented) system, more intense oxidative stress was induced by a higher concentration of nicotine in tobacco wastewater, and excessive oxidative stress was induced by 250 mg/l of nicotine at the final stage. However, when a nicotine-degrading bacterial strain Pseudomonas sp. HF-1 was added to the BA (bio-augmented) system, the oxidative stress was significantly reduced compared to the non-BA system (p < 0.01).These results suggested that the oxidative stress was mainly induced by nicotine in the SBR treatment of tobacco wastewater, and that bioaugmentation with strain HF-1 would be a potential strategy to reduce the oxidative stress and thereby improve the performance in SBRs. Additionally, the positive correlation between the nicotine content and CAT, GSH and MDA activity in both systems implied that these parameters can be used as biomarkers for reflecting the performance of SBR treatment of tobacco wastewater, and in monitoring nicotine environmental pollution.

Novibacillus thermophilus gen. nov., sp. nov., a Gram-staining-negative and moderately thermophilic member of the family Thermoactinomycetaceae Two Gram-staining-negative, facultatively anaerobic bacterial strains, SG-1 T and SG-2, were isolated from a saline soil sample and a compost sample, respectively. The cells were nonmotile rods that occurred singly or in chains, and endospores were not observed under tested growth conditions. Optimum growth occurred at 50 8C, pH 7.5-8.0 and with 5-7 % (w/v) NaCl. The DNA G+C content was 49.5-50.5 mol%. The strains contained MK-7 as the predominant menaquinone and iso-C 15 : 0 and anteiso-C 15 : 0 as the major fatty acids. The polar lipids consisted mainly of diphosphatidylglycerol and phosphatidylglycerol. The cell-wall peptidoglycan type was A1c (meso-DAP direct). Phylogenetic analyses revealed that the new isolates belonged to the family Thermoactinomycetaceae, exhibiting low 16S rRNA gene sequence similarity (90.8-91.3 %) to the nearest type strain, Mechercharimyces asporophorigenens YM11-542 T , and formed a well-supported lineage that was clearly distinguished from all currently described genera in this family. Based on our polyphasic taxonomic characterization, we propose that strains SG-1 T and SG-2 represent a novel genus and species within the family Thermoactinomycetaceae, for which we propose the name Novibacillus thermophilus gen. nov., sp. nov. The type strain of Novibacillus thermophilus is SG-1 T (5KCTC 33118 T 5CGMCC 1.12771 T ).Within the phylum Firmicutes, as the type order of the class Bacilli, the order Bacillales contains a morphologically diverse assemblage of unicellular bacteria that includes aerobic, aerotolerant, facultatively anaerobic and strictly anaerobic strains, growing under psychrophilic, mesophilic or thermophilic conditions. Members of this order are generally Gram-staining-positive and endospore-forming. As an exception, the species Bacillus horti was detected to be Gram-staining-negative, possessing a thicker peptidoglycan layer than Gram-negative bacteria but a thinner one than ordinary Gram-staining-positive bacteria (Yumoto et al., 1998). Endospore formation has not been observed under tested growth conditions for some species, such as Bacillus foraminis ( The family Thermoactinomycetaceae in the order Bacillales was proposed by Matsuo et al. (2006), consisting of the genera Laceyella, Thermoflavimicrobium, Thermoactinomyces, Seinonella, Planifilum and Mechercharimyces. At the time of writing (April 2015), with seven new genera, Desmospora, Kroppenstedtia, Lihuaxuella, Marininema, Melghirimyces, Polycladomyces and Shimazuella, the family comprised 13 recognized genera. These bacteria are characterized by the formation of a single, non-stalked spore on the aerial or substrate hyphae, or consecutive spores on straight or branched sporephores (Tsubouchi et al., 2013). Most species of the family are thermophilic, although several members of the genera Seinonella, Mechercharimyces and Shimazuella are mesophilic. In this study, we repor...