Characterization ofClostridium bifermentans and its biotransformation of 2,4,6-trinitrotoluene (TNT) and 1,3,5-triaza-1,3,5-trinitrocyclohexane (RDX)

Summary We have determined that an organism able to degrade both RDX and TNT in a pure culture is a strain ofClostridium bifermentans. The consortium from which this organism is derived also degrades these compounds, and we suspect thatC. bifermentans is also the responsible organism within that consortium. The bioconversion of RDX and TNT occurs under anaerobic conditions both in the consortium and in pure culture without the need of an added reductant. The presence of co-metabolites speeded these biotransformations.     

Pressure effects on<Emphasis Type="Italic"> Clostridium</Emphasis> strains isolated from a cold deep-sea environment

Three Clostridium strains were isolated from deep-sea sediments collected at a depth of 6.3–7.3 km in the Japan Trench. Physiological characterization and 16S rDNA analysis revealed that the three isolates were all closely related to Clostridium bifermentans. The spores of all three isolates were resistant to inactivation at high pressure and low temperature. However, despite the fact that the vegetative cells were halotolerant and eurythermal they did not appear to be adapted for growth or viability under the conditions prevailing in the deep-sea sediments from which they were obtained. The results suggest that the isolates had survived as spores in the deep-sea sediments and that the marine benthos could be a source of clostridia originating in other environments.Communicated by K. Horikoshi     

Distribution of Clostridial cry-Like Genes Among Bacillus thuringiensis and Clostridium Strains

The presence of two cry-like genes first identified in Clostridium bifermentans subsp. malaysia CH18 was investigated in Clostridium species including 12 subspecies of Clostridium bifermentans, 13 strains of other members of Clostridia genus, and 13 different subspecies of Bacillus thuringiensis. Oligonucleotides designed to amplify the two toxin genes, cmb71 and cmb72, were used. We found that these genes are present in 80% of the Clostridium bifermentans strains tested and in 8% of the other Clostridium and Bacillus thuringiensis strains. Received: 22 July 1997 / Accepted: 15 October 1997     

Partial inactivation of the mosquitocidal activity of Clostridium bifermentans serovar malaysia by extracellular proteinases

Summary Clostridium bifermentans serovar malaysia is toxic to mosquito larvae. During large-scale preparation in a fermentor, the bacteria enter the sporulation stage after 5 h culture, whereupon high larvicidal activity is obtained (LC₅₀ 48 h on Anopheles stephensi = 3.1 × 10^–5). The toxicity becomes maximal around 3–5 h later (LC₅₀ 48 h = 1.3 × 10^–5) and remains unchanged until sporangium lysis. An important loss of toxicity is then observed when the cells lyse. This loss appears to be due to the fact that C. bifermentans serovar malaysia synthesizes and excretes, mainly during vegetative growth, metallo- and/or cystein-proteinases, which are active between pH 6.0 and pH 8.0. Extracellular proteinases are most likely responsible in large part for the decrease in toxic activity concomitant with cell lysis. Lysis is however prevented by addition of 10 mM ethylenediaminetetraacetic acid to the culture medium before forespore formation, and under these conditions the larvicidal activity can be maximized. Offprint requests to: L. Nicolas     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.     

Sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, as determined by PCR amplification procedure

Aims: To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum‐packed chilled meats. Methods and Results: Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb‐processing plants, their environments, dressed carcasses and final vacuum‐packed meat stored at –0·5°C for 5½ weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro‐organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. Conclusions: The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for ‘blown pack’ ‐causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. Significance and Impact of the Study: This paper provides information significant for controlling meat spoilage‐causing clostridia in the meat‐processing plants.     

Quantitative Chemical Analyses and Antigenic Properties of Peptidoglycans from Clostridium botulinum and Other Clostridia

The cell wall peptidoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The peptidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histolyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the Clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other Clostridia as well as various types of C. botulinum.     

Characterization of enolase fromClostridium difficile

We have isolated and purified enolase fromClostridium difficile. This is the first report of an enolase of theClostridium genus, and in general its characteristics resemble those described previously for other species, except that it is extremely thermostable. Interestingly,C. difficile enolase has an octameric structure (approximately 300 kDa on native PAGE, 50 kDa on SDS PAGE, and 338 kDa by gel filtration). Enolases fromC. sordellii andC. bifermentans have been partially purified and have a molecular weight similar to that ofC. difficile. It may be that this large size is common for enolases isolated from bacteria of theClostridium genus.     

Inorganic nitrogen metabolism in two cellulose-degrading clostridia

Inorganic nitrogen metabolism in two cellulose degrading clostridia, the mesophile Clostridium cellobioparum and the thermophile Clostridium thermocellum was investigated. Both strains show acetylene reduction (i.e. possibly nitrogenase activity), contain glutamine synthetase, glutamate dehydrogenase and glutamate-dependent transaminases. C. cellobioparum additionally contains a NADH-dependent glutamate synthase and a NH ₄ ⁺ -repressible glycine dehydrogenase (NADPH). Remarkably, acetylene reduction in C. thermocellum is not repressed by ammonium, casting doubt whether this activity is due to nitrogenase. The results are compared with the data from other saccharolytic clostridia.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase (glutamate synthase)     

Intrinsic factors in meat products counteracting botulinogenic conditions

Spores ofClostridium botulinum occur in soil, dung and dirt, materials which are normally present on the skin of living pigs entering the slaughter-house. After slaughtering, the surface of the pigs may become contaminated with these materials, the chances and the level of contamination depending on the hygienic conditions in the factory.In vacuum-packed pre-cooked meat products anaerobic conditions prevail, which may allow the development of clostridia. As an example we have studied a typical Dutch smoked ring sausage: Gelderse rook-worst. The press-juice of this sausage was used as the culture medium to investigate the influence of temperature (25–37 C), of pH (5.7–7.0), of salt (2.5–5.0%) and of nitrite (0.005–0.05%) on the development ofClostridium botulinum type B, ATCC strain No. 438. Germination of spores and growth was inhibited for 4 weeks at 30 C by a combination of pH<5.8, salt>4.5% and nitrite>0.05%.These intrinsic factors in a meat product (pH, salt and nitrite) decide on germination of spores and growth ofClostridium botulinum and determine the safety of vacuum-packed products.     

Improving the <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol fermentation by engineering the strain for co-production of riboflavin

Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli–Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.     

Comparative studies on physiology and taxonomy of obligately purinolytic clostridia

Eleven strains of obligately purinolytic clostridia have been studied with respect to their assignment to the three type strains of Clostridium acidiurici, C. cylindrosporum, and C. purinolyticum. DNA/DNA-hybridization proved to be the method of choice for differentiation whereas phenotypic characteristics such as spore morphology, substrate spectra, nutritional requirements, product formation, and sensitivity against various antibiotics did not allow unequivocal identification. All strains depended on selenite for growth.     

Physiological characteristics ofClostridium bifermentans selectively isolated from California desert tortoise

A selective agar medium containing cycloserine (250 mg/L), sulfamethoxazole (76 mg/L), and trimethoprim (4 mg/L) was used for isolation ofClostridium bifermentans from the intestinal contents of California desert tortoise. Typical lecithinase positive colonies that appeared on the plates, were biochemically characterized with the API 20A System and a conventional procedure. The susceptibility of the isolates to 12 antimicrobial agents was determined by the broth microtitration technique using the ANA MIC System.C. bifermentans strains were shown to be highly susceptible to cefoxitin, cefotetan, ceftizoxime, cefotaxime, chloramphenicol, clindamycin, penicillin, metronidazole, piperacillin, ticarcillin, and mezlocillin. Less than 10% of the isolates exhibited resistance to tetracycline. All strains were found to be highly resistant to cycloserine, sulfamethoxazole, and trimethoprim. The GLC analysis of the culture supernatants for volatile fatty acids revealed the presence of formic, acetic, isobutyric, butyric, isovaleric, and isocaproic acids.     

Microbial interactions associated with secondary cucumber fermentation

Aims

To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation.

Methods and Results

Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4·6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3·2), but only E. cloacae was able to produce a secondary product.

Conclusions

The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids.

Significance and Impact of the Study

Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.     

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO₂-gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.     

CRISPR‐based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii

Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all‐in‐one, CRISPR‐based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target‐specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene‐specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9‐mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR‐Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia.     

Characterization of 10 mesophilic cellulolytic clostridia isolated from a municipal solid waste digestor

By hybridization experiments with three cloned fragments carrying cellulase genes ofClostridium cellulolyticum, we tried to differentiate 10 cellulolytic mesophilic clostridia, isolated from a municipal solid waste digestor. On the basis of hybridization experiments, three major groups were found among the 10 isolates. The two endoglucanase genes,cel CCA andcel CCB ofC. cellulolyticum, hybridized with nine strains of our isolates, suggesting homology and widespread distribution of these genes. Withcel CCA the strain A31 exhibited a different pattern. In contrast to these nine strains, the strain A11 was found to share no or very weak homology with these two probes, which indicated that this strain of cellulolytic clostridia possesses nonidentical cellulase complex. None of these new strains hybridized withnif genes, indicating that these clostridia did not appear to be nitrogen-fixing bacteria. With other biochemical characteristics, we found that these bacteria appeared to be different from the presently known mesophilic cellulolytic clostridia.     

Competition for L-glutamate between specialised and versatile Clostridium species

Clostridium cochlearium could be reproducibly enriched in an L-aspartate- and L-glutamate-limited, anaerobic chemostat inoculated with anaerobic sludge. L-glutamate, L-glutamine and L-histidine were the only fermentable substrates. Less specialised clostridia of the C. tetanomorphum type could only be isolated from batch enrichments with L-glutamate and L-aspartate as energy sources. Competition experiments with C. cochlearium and C. tetanomorphum in a L-glutamate-limited chemostat resulted in the selective elimination of the latter species. Addition of glucose to the medium resulted in coexistence of both species. The molar growth yields for L-glutamate at different dilution rates at 30°C were determined for both species. The maximum specific growth rates on L-glutamate were 0.55 h^-1 for C. cochlearium and 0.35 h^-1 for C. tetanomorphum.