Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. SIGNIFICANCE AND IMPACT OF THE STUDY: There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future.

Effect of electro-activated aqueous solutions, nisin and moderate heat treatment on the inactivation of Clostridium sporogenes PA 3679 spores in green beans puree and whole green beans.

In this work, the synergistic effect of electro-activated solutions (EAS) of potassium acetate and potassium citrate, nisin and moderate heat treatment to inactivate C. sporogenes PA 3679 spores was evaluated in green beans puree and whole green beans. Electro-activated solutions (EAS) of potassium acetate and potassium citrate were generated under 400 mA during 60 min. They were characterized by an oxidation-reduction potential (ORP) and pH values ranged from +300 to +1090 mV and 2.8 to 3.67, respectively. Moreover, the EAS were combined with a bacteriocin nisin at concentrations of 250, 500, 750 and 1000 IU/mL and the targeted sporicidal effect was evaluated under moderate heat treatment. The inoculated mixtures were subjected to temperatures of 95, 105 and 115 °C for exposure times of 5, 15 and 30 min. After plate counting, the synergistic effect of the hurdle principle composed of electro-activated solutions, nisin and moderate temperatures was demonstrated. The obtained results showed that the synergistic effect of the used hurdle was able to achieve an inactivation efficacy of 5.9-6.1 log CFU/mL. Furthermore, experiments carried out with whole green beans showed that spore inactivation level was significantly higher and reach 6.5 log CFU/mL. Moreover, spore morphology was examined by transmission electron microscopy and the obtained micrographs showed important damages in all of the treated spores.

Combined effects of marinating and γ-irradiation in ensuring safety, protection of nutritional value and increase in shelf-life of ready-to-cook meat for immunocompromised patients.

The aim of this study was to evaluate the effect of combining marinating and γ-irradiation at doses of 1, 1.5 and 3kGy on Escherichia coli O157:H7, Salmonella typhimurium and Clostridium sporogenes in raw meat packed under vacuum and stored at 4°C and to estimate its safety and shelf-life. Further, the effect of combined treatments on sensorial, nutritional values (lipid oxidation, concentration of thiamin and riboflavin) and color was evaluated. The study demonstrated that the use of marinade in combination with a low dose of γ-irradiation (1.5kGy) could act in synergy to reduce to undetectable level of pathogenic bacteria and increase the shelf-life of ready-to-cook meat loin without affecting its sensorial and nutritional quality.

Study of the combined effect of electro-activated solutions and heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model solution and vegetable puree.

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Spore Resistance Properties.

Spores of various Bacillus and Clostridium species are among the most resistant life forms known. Since the spores of some species are causative agents of much food spoilage, food poisoning, and human disease, and the spores of Bacillus anthracis are a major bioweapon, there is much interest in the mechanisms of spore resistance and how these spores can be killed. This article will discuss the factors involved in spore resistance to agents such as wet and dry heat, desiccation, UV and γ-radiation, enzymes that hydrolyze bacterial cell walls, and a variety of toxic chemicals, including genotoxic agents, oxidizing agents, aldehydes, acid, and alkali. These resistance factors include the outer layers of the spore, such as the thick proteinaceous coat that detoxifies reactive chemicals; the relatively impermeable inner spore membrane that restricts access of toxic chemicals to the spore core containing the spore's DNA and most enzymes; the low water content and high level of dipicolinic acid in the spore core that protect core macromolecules from the effects of heat and desiccation; the saturation of spore DNA with a novel group of proteins that protect the DNA against heat, genotoxic chemicals, and radiation; and the repair of radiation damage to DNA when spores germinate and return to life. Despite their extreme resistance, spores can be killed, including by damage to DNA, crucial spore proteins, the spore's inner membrane, and one or more components of the spore germination apparatus.

"Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm⁻¹, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change.

In situ determination of Clostridium endospore membrane fluidity during pressure-assisted thermal processing in combination with nisin or reutericyclin.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

Synergistic effects of high hydrostatic pressure, mild heating, and amino acids on germination and inactivation of Clostridium sporogenes spores.

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro.

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.

Micro-organism re-growth in wastewater disinfected by UV radiation and ozone: a micro-biological study.

A series of disinfection experiments using UV radiation and ozone was performed on the secondary effluent from a wastewater treatment plant at a pilot plant scale. The microbial population in the inflowing wastewater and the treated outflow water were quantified for each of the treatment modules (fecal coliforms, fecal streptococci, Salmonella spp. (presence/absence), Clostridium Sulphite-reducers, Pseudomonas aeruginosa, Staphylococcus aureus, coliphages, nematodes, intestinal nematodes and pathogenic fungi). Treated water was stored in opaque tanks at a temperature between 20 and 22 degrees C, after which, a one-month study of the regrowth of the bacterial flora, nematodes and fungi was carried out. Clostridium Sulphite-reducers, pathogenic fungi and nematodes were the micro-organisms showing a greatest degree of resistence to UV- and Ozone-treatment. It was only concerning Clostridium and Pseudomonas abatement that significant elimination results were achieved with both technologies.

Producing hydrogen from wastewater sludge by Clostridium bifermentans.

Excess wastewater sludge collected from the recycling stream of an activated sludge process is biomass that contains large quantities of polysaccharides and proteins. However, relevant literature indicates that the bio-conversion of wastewater sludge to hydrogen is limited and therefore not economically feasible. This work examined the anaerobic digestion of wastewater sludge using a clostridium strain isolated from the sludge as inoculum. A much higher hydrogen yield than presented in the literature was obtained. Also, the effects of five pre-treatments-ultrasonication, acidification, sterilization, freezing/thawing and adding methanogenic inhibitor-on the production of hydrogen were examined. Freezing and thawing and sterilization increased the specific hydrogen yield by 1.5-2.5 times to that of untreated sludge, while adding an inhibitor and ultrasonication reduced the hydrogen yield.

Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters.

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.

Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation.

One of the major problems with gene therapy today is the lack of tumour specificity. The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour. These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour. As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter. A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy. A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05). These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter. Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression. Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

Comparative effects of gamma and microwave irradiation on the quality of black pepper.

Powdered black pepper from Egyptian markets, was irradiated with different recommended doses of gamma rays (5.0 and 10.0 kGy) and with microwaves for different periods (20, 40 and 75 s) to improve its hygienic quality. The most common bacterial isolates were of three genera Bacillus, Clostridium and Micrococcus (7.5 x 10(6)), whereas the predominant fungi (7.8 x 10(4)) were Aspergillus species, A. glaucus, A. flavus, A. niger and A. ochraceus. Doses of gamma irradiation used (5.0 and 10 kGy) were sufficient to decrease spore-forming bacteria (SFB) and to inhibit the fungal flora and coliforms which contaminated the black pepper powder. Microwave treatments for 40 s and 75 s were of the same effectiveness whereas treatment for 20 s was less so. GLC analysis proved the presence of 31 peaks, only 19 compounds were identified as monoterpene hydrocarbons (56.21%), the major one being beta-phellandrene and limonene. Sesquiterpenes were also present, mainly beta-caryollphyllene (3.69%) as well as oxygenated compounds such as terpenol, geraniol, Me-chavicol, eugenol and anisol. Gamma irradiation at 5 kGy and 10 kGy respectively decreased the numbers of identified compounds from 21 (86.58% concentration) in untreated pepper to 16 (59.22% concentration), 15 (54.06% concentration). In comparison, microwave treatments, particularly for 40 s and 75 s, increased the concentration of the same compounds. The results obtained indicate that microwave treatment, under these conditions, is a safe and suitable technique for decontamination of black pepper which does not result in a great loss of flavour compounds, as compared with recommended doses of gamma irradiation.

Effect of microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores.

Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating.

Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers.

UV irradiation of complexes of DNA and an alpha/beta-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were less than 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m2; in the absence of SASP the yields were reversed-4.5% and 0.3%, respectively. Complexes of DNA with alpha/beta-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of alpha/beta-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.

Mutagenesis of Clostridium acetobutylicum.

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N-methyl-N'-nitro-N-nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.