We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

The solventogenic sol operon consisting of bld, ctfA, ctfB, and adc was cloned from Clostridium saccharoperbutylacetonicum strain N1-4. These genes share as high as 95-98% similarity with the corresponding sol genes of Clostridium beijerinckii NCIMB 8052. The N1-4 sol gene cluster was transcribed in a polycistronic manner under the control of two promoters, and its transcription was highly induced during solventogenesis. Strain DGN3-4, the degenerated strain derived from N1-4, maintained the sol genes, but transcription of the DGN3-4 sol operon was hardly induced during solventogenesis. A substance extracted from the culture supernatants of wild-type N1-4 allowed us to induce transcription of the sol operon in DGN3-4. These results suggest that the degeneration is caused by the incompetence of the induction mechanism of the sol operon, and that transcription might be under the control of a quorum-sensing mechanism.

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 M, but not carbon dichloride even at 1 mM.Chlorinated organic solvents, such as tetrachloroethene (PCE) and trichloroethene (TCE), have been recognized worldwide as some of the most serious environmental pollutants. Because highly chlorinated chemicals are typically not degraded through oxygenation by aerobic bacteria, anaerobic dehalorespiring bacteria have received increased attention in the last decade. Dehalorespiration is an efficient dechlorination mechanism in which halogenated compounds can be used as the final electron acceptor. This reductive dechlorination is coupled with energy-yielding phosphorylation. Dehalorespiring bacteria are believed to play an important role in the degradation of chlorinated environmental pollutants. Since Desulfomonile tiedjei DCB-1, capable of the reductive dechlorination of chlorobenzoates, was first reported (5, 16), a number and variety of dehalorespiring bacteria have been isolated to date (4,10,11,13,17,18,22). Desulfitobacterium hafniense strain Y51 is one of these bacteria that dechlorinates PCE to cis-1,2-dichloroethene (cis-DCE) via TCE (19).We previously purified and characterized the reductive PCE dehalogenase PceA and cloned the corresponding pceA gene from Desulfitobacterium hafniense strain Y51 (GenBank accession no. AY706985) (20). Subsequently, the rest of the pce genes, such as pceB, pceC, and pceT, were cloned (GenBank accession no. AY706985) (7,20). The pceABCT gene cluster was situated between the two nearly identical IS elements termed ISDesp1 and ISDesp2, which belong to the IS256 family. Thus, the set of these genes could form a composite transposon (8). We isolated two different PCE-nondechlorinating variants that emerged during repeated subculturing of strain Y51 and named them SD (small deletion) and LD (large deletion) (8). Sequencing analysis revealed that the SD variant lost the ISDesp1 element (ca. 1.6-kb DNA region upstream of pceA), which includes the Ϫ35 promoter sequence of the pceA gene, so that the SD variant failed to transcribe the pceA gene. On the other han...

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The selective production of acetone and butanol is highly desirable from the viewpoint of biofuel production. We have manipulated the activity level of a hydrogenase for this purpose because hydrogen and solvent production are closely correlated with each other. First, we cloned the hydrogenase gene cluster from Clostridium saccharoperbutylacetonicum strain N1-4 and downregulated its expression using an antisense RNA strategy. The cloned hydrogenase gene cluster contained three adjacent open reading frames, designated hupC, hupB, and hupA. Sequence analysis revealed that HupA could accommodate an H-cluster, which is the catalytic domain of the Fe-hydrogenase. HupB and HupC contained no H-cluster but could accommodate several Fe-S clusters. The hupCBA genes were co-transcribed, and the level of the transcript was maximized in the solventogenic phase. When the antisense RNA of the hupC upstream region (180 bp) was expressed under the bdh (encoding butanol dehydrogenase) promoter, significant reduction of hupC translation was observed, indicating that this antisense RNA is effective in strain N1-4. Production of hydrogen in the antisense transformant increased 3.1-fold. Hydrogen-evolving activity was comparable in both the control and antisense strains, but hydrogen uptake activity significantly decreased in the antisense strain (13% remaining). These results indicate that the HupCBA proteins are involved in hydrogen uptake. Importantly, the level of acetone in the antisense transformant increased 1.6-fold, and butanol production decreased to 75.6% compared to the control strain. Thus, we successfully altered solvent productivity by controlling electron flow in an acetone/butanol-producing Clostridium species.