Methylmercury Resistance in Desulfovibrio desulfuricans Strains in Relation to Methylmercury Degradation

Two strains of Desulfovibrio desulfuricans, one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of Clostridium pasteurianum, a H₂ producer, and with the broad-spectrum mercury-resistant Pseudomonas putida strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH₃HgCl resistance of D. desulfuricans strains was 10 times that of P. putida FB-1 and 100 times that of C. pasteurianum. The methylmercury resistance of D. desulfuricans was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH₃)₂Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH₃HgCl. This was compared with a 1-liter culture of D. desulfuricans LS spiked with 100 mg of HgCl₂. In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH₃Hg)₂S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The D. desulfuricans strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter^-1) or low (0.09-g · liter^-1) sulfate contents. Methane was produced from CH₃HgCl at a lower rate than by the broad-spectrum Hg-resistant P. putida strain FB-1.     

Methane oxidation and methane driven redox process during sequential reduction of a flooded soil ecosystem

A laboratory incubation study conducted to assess the temporal variation of CH₄ oxidation during soil reduction processes in a flooded soil ecosystem. A classical sequence of microbial terminal electron accepting process observed following NO₃ ^? reduction, Fe³⁺ reduction, SO₄ ^2? reduction and CH₄ production in flooded soil incubated under initial aerobic and helium-flushed anaerobic conditions. CH₄ oxidation in the slurries was influenced by microbial redox process during slurry reduction. Under aerobic headspace condition, CH₄ oxidation rate (k) was stimulated by 29 % during 5 days (NO₃ ^? reduction) and 32 % during both 10 days (Fe³⁺) and 20 days (early SO₄ ^2? reduction) over unreduced slurry. CH₄ oxidation was inhibited at the later methanogenic period. Contrastingly, CH₄ oxidation activity in anaerobic incubated slurries was characterized with prolonged lag phase and lower CH₄ oxidation. Higher CH₄ oxidation rate in aerobically incubated flooded soil was related to high abundance of methanotrophs (r?=?0.994, p?<?0.01) and ammonium oxidizers population (r?=?0.184, p?<?0.05). Effect of electron donors NH₄ ⁺, Fe^2+, S^2? on CH₄ oxidation assayed to define the interaction between reduced inorganic species and methane oxidation. The electron donors stimulated CH₄ oxidation as well as increased the abundance of methanotrophic microbial population except S^2? which inhibited the methanotrophic activity by affecting methane oxidizing bacterial population. Our result confirmed the complex interaction between methane-oxidizing microbial groups and redox species during sequential reduction processes of a flooded soil ecosystem.     

Structure–activity relationships of lipopolysaccharide sequestration in N-alkylpolyamines

We have previously shown that simple N-acyl or N-alkyl polyamines bind to and sequester Gram-negative bacterial lipopolysaccharide, affording protection against lethality in animal models of endotoxicosis. Several iterative design-and-test cycles of SAR studies, including high-throughput screens, had converged on compounds with polyamine scaffolds which have been investigated extensively with reference to the number, position, and length of acyl or alkyl appendages. However, the polyamine backbone itself had not been explored sufficiently, and it was not known if incremental variations on the polymethylene spacing would affect LPS-binding and neutralization properties. We have now systematically explored the relationship between variously elongated spermidine [NH₂–(CH₂)₃–NH–(CH₂)₄–NH₂] and norspermidine [NH₂–(CH₂)₃–NH–(CH₂)₃–NH₂] backbones, with the N-alkyl group being held constant at C₁₆ in order to examine if changing the spacing between the inner secondary amines may yield additional SAR information. We find that the norspermine-type compounds consistently showed higher activity compared to corresponding spermine homologues.     

Mechanism of toxicity of platinum(II) compounds in repair-deficient strains of Escherichia coli

The survival of wild-type and repair-deficient Escherichia coli treated with cis-Pt(NH₃)₂Cl₂, trans-Pt(NH₃)₂Cl₂ and [Pt(dien)Cl]Cl (dien = H₂NCH₂CH₂NHCH₂CH₂NH₂) was inversely correlated with the ability of these compounds to inhibit DNA synthesis in different bacterial strains. The relative amounts of these 3 compounds covalently bound to DNA immediately after treatment with the same dose were, respectively, 1:?2:1, their relative abilities to inhibit DNA synthesis were 6:1:0 and their relative toxicities toward the wild-type and uvrA strains were 3–5:1:0. More repair synthesis, as measured by density-gradient centrifugation techniques, was observed in wild-type bacteria after treatment with the cis than with the trans isomer whereas no repair synthesis was detected after exposure to [Pt(dien)Cl]Cl.These results are consistent with the hypothesis that cis-Pt(NH₃)Cl₂ binds to DNA and inhibits DNA synthesis thereby killing the cell. The lower toxicity of this compound toward wild-type bacteria compared with repair-deficient strains is in part a consequence of DNA repair. trans-Pt(NH₃)₂Cl₂ and [Pt(dien)Cl]Cl are less toxic than the cis isomer; this lesser toxicity is not a consequence of low levels of DNA binding or enhanced repair of the lesions but appears to reflect a weaker inhibition of DNA synthesis by these Pt-DNA adducts.     

Identification of biogenic dimethyl selenodisulfide in the headspace gases above genetically modified Escherichia coli

Escherichia coli JM109 cells were modified to express the genes encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant thermophile, Geobacillus stearothermophilus V. Manual headspace extraction was used to collect the gases for gas chromatography with fluorine-induced sulfur chemiluminescence analysis while solid-phase microextraction was used for sample collection in gas chromatography/mass spectrometry (GC/MS) analysis. When grown in the presence of selenate or selenite, these bacteria produced both organo-sulfur and organo-selenium in the headspace gases above the cultures. Organo-sulfur compounds detected were methanethiol, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Organo-selenium compounds detected were dimethyl selenide and dimethyl diselenide. Two mixed sulfur-selenium compounds, dimethyl selenenyl sulfide and a chromatographically late-eluting compound, were detected. Dimethyl selenodisulfide, CH(3)SeSSCH(3), and dimethyl bis(thio)selenide, CH(3)SSeSCH(3), were synthesized and analyzed by GC/MS and fluorine-induced chemiluminescence to determine which corresponded to the late-eluting compound that was bacterially produced. CH(3)SeSSCH(3) was positively identified as the compound detected in bacterial headspace above Se-amended cultures. Using GC retention times, the boiling point of CH(3)SeSSCH(3) was estimated to be approximately 192 degrees C. This is the first report of CH(3)SeSSCH(3) produced by bacterial cultures.     

Disparity in productive binding mode of the slow-reacting enantiomer determines the novel catalytic behavior of Candida antarctica lipase B

In the context of specifying the origin of enzyme enantioselectivity, the present study explores the lipase enantioselectivity towards secondary alcohols of similar structure from the perspective of substrate binding. By carrying out molecular mechanics minimization as well as molecular dynamics simulation on tetrahedral reaction intermediates which are used as a model of transition state, we identify an unconventional productive binding mode (PBM)—M/H permutation type for Candida antarctica lipase B (CALB). The in silico results also indicate that different PBMs of the slow-reacting enantiomer do exist in one lipase even when there is little structural differences between substrates, e.g. compounds with Ph or CH₂CH₂Ph group display the M/H permutation type PBM while molecules with CH₂Ph show the M/L permutation type PBM. By relating the PBMs of substrates to the experimentally determined E-values obtained by Hoff et al. [16], we find that disparity in PBM of the slow-reacting enantiomer determines why E-values of substrates with CH₂Ph were lower than E-values of substrates with Ph or CH₂CH₂Ph group. The modeling results also suggest that the “pushed aside” effect of the F atom and Br atom accommodates the medium size substituent of the substrate better in the stereospecificity pocket of the enzyme.     

Chloromethane, a Novel Methyl Donor for Biosynthesis of Esters and Anisoles in Phellinus pomaceus

Chloromethane (CH₃Cl), a gaseous natural product released as a secondary metabolite by many woodrotting fungi of the family Hymenochaetaceae, has been shown to act as a methyl donor for biosynthesis of methyl esters of benzoic and furoic acid in the primary metabolism of Phellinus pomaceus. The broad-specificity methylating system could esterify a wide range of aromatic and aliphatic acids. In addition to CH₃Cl, both bromo- and iodomethanes acted as methyl donors. Methylation did not appear to proceed via methanol or a coenzyme A intermediate. The initial growth-related accumulation of methyl benzoate during culture of P. pomaceus was paralleled by an increase in activity of the methylating system in the mycelium. Changes in percent incorporation of C²H₃ from exogenous C²H₃Cl during growth indicated that although utilization of CH₃Cl was initially closely coupled to biosynthesis of the compound, the system became less tightly channeled later in growth. This phase coincided with release of gaseous CH₃Cl by the fungus. A biochemically distinct CH₃Cl-utilizing system capable of methylating phenols and thiophenol was also identified in the fungus, but in contrast with the carboxylic acid-methylating system, it attained maximum activity in the idiophase. Preliminary investigations of a non-CH₃Cl-releasing fungus, Fomitopsis pinicola, have shown the presence of a CH₃Cl-utilizing system capable of methylating benzoic acid, suggesting that CH₃Cl biosynthesis may occur in non-hymenochaetaceous fungi. Halogenated compounds hitherto found in nature are mainly stable end products of metabolism. The participation of CH₃Cl in primary fungal metabolism demonstrates that some halometabolites may have a previously unrecognized role as intermediates in the biosynthesis of nonhalogenated natural products.     

Assessing the effects of chamber placement, manual sampling and headspace mixing on CH4 fluxes in a laboratory experiment

A laboratory experiment was conducted with two types of closed static chambers to estimate the effects of chamber placement, manual headspace sampling and headspace mixing on methane (CH₄) fluxes. Chamber fluxes were compared to a known reference flux in a chamber calibration system. The measurements were conducted with three types of soils (coarse dry, fine dry and fine wet quarts sand) at five flux levels ranging from 60 to 2000 ??g CH₄ m^?2 h^?1. We found that the placement of a non-vented chamber disturbed the initial CH₄ concentration development within the chamber headspace for 10 to 30 s. Excluding this short period from the flux calculation resulted in a lower flux estimate (mean±SE) of 126?±?26 ??g CH₄ m^?2 h^?1 compared to 134?±?26 ??g CH₄ m^?2 h^?1 if data from time zero of the enclosure were included. We also found that in non-mixed chambers (no fan mixing) the gas sampling by syringes or gas bottles disturbed the development of CH₄ concentration during the enclosure. Furthermore, flux estimates in non-mixed chambers were significantly underestimated (on average 36%) compared to the measured reference fluxes. However, the use of fans to constantly mix the chamber headspace during enclosure significantly improved the goodness-of-fit of the regression analysis used to calculate the flux and further eliminated the disturbance of the manual sampling on the concentration development. We recommend that chambers should be vented during the placement of the chamber, and that fans are used as an integrated part of static chambers while headspace mixing with syringes should be avoided.     

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H₂ to the culture headspace had the most significant positive effect on the growth yield under CH₄ (6%) and O₂ (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH₄, while CH₄ consumption was significantly reduced. Regardless of H₂ addition, CH₄ utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O₂/CH₄ mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H₂ addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H₂ (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H₂ may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH_4, such as the production of high-quality feed protein.     

End Products of Anaerobic Chitin Degradation by Salt Marsh Bacteria as Substrates for Dissimilatory Sulfate Reduction and Methanogenesis

The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other salt marsh bacteria. Actively growing chitinoclastic bacterial isolates produced primarily acetate, H₂, and CO₂ in broth culture. No sulfate-reducing or methanogenic isolates grew on chitin as sole carbon source or produced any measurable degradation products. Mixed cultures of chitin degraders with sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin-degrading isolates with methanogens resulted in the production of CH₄ with reductions in headspace CO₂ and H₂. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide, with more methane being produced in mixed cultures containing CO₂-reducing methanogens and acetoclastic sulfate reducers because of less interspecific H₂ competition.     

Increase of fungitoxicity of mercuric chloride by methionine,ethionine and S-methylcysteine

The fungitoxicity of mercuric chloride to Aspergillus niger was increased in the presence of d-, l-, dl-methionine, dl-ethionine, dl-S-methylcysteine or sodium methylmercaptide. The same effect was observed with methionine for two other fungi investigated: Cladosporium cucumerinum and Scopulariopsis brevicaulis. It is suggested that this effect can be ascribed to the formation of CH₃SHg⁺ or (CH₃S)₂Hg, or the corresponding ethyl compounds. CH₃SHgCl and (CH₃S)₂Hg were synthetically prepared and proved indeed far more fungitoxic than HgCl₂. The hypothesis was further substantiated by the observation that A. niger rapidly converts dl-methionine into CH₃SH, which undoubtedly reacts with Hg²⁺ to give the above mentioned methylthiomercury compounds.     

Improved enrichment culture technique for methane-oxidizing bacteria from marine ecosystems: the effect of adhesion material and gas composition

Cultivation of microbial representatives of specific functional guilds from environmental samples depends largely on the suitability of the applied growth conditions. Especially the cultivation of marine methanotrophs has received little attention, resulting in only a limited number of ex situ cultures available. In this study we investigated the effect of adhesion material and headspace composition on the methane oxidation activity in methanotrophic enrichments obtained from marine sediment. Addition of sterilized natural sediment or alternatively the addition of acid-washed silicon dioxide significantly increased methane oxidation. This positive effect was attributed to bacterial adhesion on the particles via extracellular compounds, with a minimum amount of particles required for effect. As a result, the particles were immobilized, thus creating a stratified environment in which a limited diffusive gas gradients could build up and various microniches were formed. Such diffusive gas gradient might necessitate high headspace concentrations of CH₄ and CO₂ for sufficient concentrations to reach the methane-oxidizing bacteria in the enrichment culture technique. Therefore, high concentrations of methane and carbon dioxide, in addition to the addition of adhesion material, were tested and indeed further stimulated methane oxidation. Use of adhesion material in combination with high concentrations of methane and carbon dioxide might thus facilitate the cultivation and subsequent enrichment of environmentally important members of this functional guild. The exact mechanism of the observed positive effects on methane oxidation and the differential effect on methanotrophic diversity still needs to be explored.     

Proton Transfer Reaction Mass Spectrometry detects rapid changes in volatile metabolite emission by Mycobacterium smegmatis after the addition of specific antimicrobial agents

The metabolic activity of plants, animals or microbes can be monitored by gas headspace analysis. This can be achieved using Proton Transfer Reaction Mass Spectrometry (PTR-MS), a highly sensitive detection method for trace gas analysis. PTR-MS is rapid and can detect metabolic responses on-line as they occur. Here, we study the headspace of actively growing cultures of paired ciprofloxacin sensitive and resistant bacterial strains (Mycobacterium smegmatis in Middlebrook M7H9 liquid media) after the addition of the antibiotics ciprofloxacin and gentamicin in real time. Following the emission patterns of the mycobacteria over time allowed volatile markers specific for the bacterial response to each antibiotic to be detected. A proportion of the measured responses were very rapid, occurring within three hours after the addition of the compounds and varied between isolates with different resistance phenotypes. Specifically, we observed a two fold increase of m73 (unidentified C4 compound) within 10 h after the addition of ciprofloxacin and a threefold increase of m45 (acetaldehyde) within 4 h after the addition of gentamicin as compared to values before the addition. Monitoring the emission of specific volatiles into the culture headspace thus has the potential for rapid drug susceptibility testing. Moreover, these and other differences in the measured responses to the two tested compounds provide evidence that monitoring multiple compounds may also give an indication of the mechanism of action of the compound added.     

A comparison of the 8-hydroxydeoxyguanosine,chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes

We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxydeoxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH₃HgCl) and mercuric chloride (HgCl₂) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH₃HgCl or HgCl₂ increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 × 10⁻⁵ M (HgCl₂) or 2 × 10⁻⁶ M (CH₃HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. CH₃HgCl was about 4-fold more potent than HgCl₂. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH₃HgCl at a concentration that was about 50% of the amount of HgCl₂ required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl₂- and/or CH₃HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.     

Transgenic Bt rice has adverse impacts on CH4 flux and rhizospheric methanogenic archaeal and methanotrophic bacterial communities

Background and Aims

The effect of transgenic insect-resistant crops on soil microorganisms has become an issue of public concern. The goal of this study was to firstly realize the variation of in situ methane (CH₄) emission flux and methanogenic and methanotrophic communities due to planting transgenic Bt rice (Bt) cultivar.

Methods

CH₄ emitted from paddy soil was collected by static closed chamber technique. Denaturing gradient gel electrophoresis and real-time PCR methods were employed to analyze methanogenic archaeal and methanotrophic bacterial community structure and abundance.

Results

Results showed that planting Bt rice cultivar effectively reduced in situ CH₄ emission flux and methanogenic archaeal and methanotrophic bacterial community abundance and diversity. Data analysis showed that in situ CH₄ emission flux increased significantly with the increase of methanogenic archaeal abundance (R ² ?=?0.839, p?<?0.001) and diversity index H′ (R ² ?=?0.729, p?<?0.05), whereas was not obviously related to methanotrophic bacterial community.

Conclusions

Our results suggested that the lower in situ CH₄ emission flux from Bt soil may result from lower methanogenic archaeal community abundance and diversity, lower methanogenic activity and higher methanotrophic activity. Moreover, our results inferred that specific functional microorganisms may be a more sensitive indicator than the total archaeal, bacterial or fungal population to assess the effects of transgenic insect-resistant plants on soil microorganisms.     

Synthesis and molecular structure of the all-trans- and the trans-cis-isomers of dichlorodimethyltin complexes of phosphoric triamides

Four new phosphoramidates with formula 4-RC₆H₄C(O)NHP(O)(NH(CH(CH₃)₂)₂, R = H (1), OCH₃ (2), CH₃ (3), Cl (4) and their diorganotin(IV) complexes with formula SnCl₂(CH₃)₂(X)₂, X = 1 (5), 2 (6), 3 (7) and 4 (8) were synthesized and characterized by NMR, IR spectroscopy and elemental analysis. The spectroscopic properties of complexes were compared with those corresponding ligands. The molecular structures for 5, 5·CH₃CN, 6·CH₃CN, 7 and 8 were established by X-ray diffraction analysis and shown that the tin atoms have a distorted octahedral coordination with trans-methyl groups. Two different all-trans and cis-trans isomers were obtained by changing the crystallization solvent system. The existence of CH₃CN in molecular packing of trans-cis isomers might be a packing factor governing the orientation of the ligands. Due to the presence of several hydrogen bond donors and acceptors on compounds, extended hydrogen-bonded networks were observed. The structure of 2 was also determined and possesses two crystallographically independent molecules in asymmetric unit. On forming complex 6·CH₃CN, the ligand 2 shows shortening of CO and P-N bond distances and increasing of PO bond length. Pseudopolymorphism of diorganotins in 5 and 5·CH₃CN is reported for the first time.     

Bacterial Oxidation of Dibromomethane and Methyl Bromide in Natural Waters and Enrichment Cultures

Bacterial oxidation of ¹⁴CH₂Br₂ and ¹⁴CH₃Br was measured in freshwater, estuarine, seawater, and hypersaline-alkaline samples. In general, bacteria from the various sites oxidized similar amounts of ¹⁴CH₂Br₂ and comparatively less ¹⁴CH₃Br. Bacterial oxidation of ¹⁴CH₃Br was rapid in freshwater samples compared to bacterial oxidation of ¹⁴CH₃Br in more saline waters. Freshwater was also the only site in which methyl fluoride-sensitive bacteria (e.g., methanotrophs or nitrifiers) governed brominated methane oxidation. Half-life calculations indicated that bacterial oxidation of CH₂Br₂ was potentially significant in all of the waters tested. In contrast, only in freshwater was bacterial oxidation of CH₃Br as fast as chemical removal. The values calculated for more saline sites suggested that bacterial oxidation of CH₃Br was relatively slow compared to chemical and physical loss mechanisms. However, enrichment cultures demonstrated that bacteria in seawater can rapidly oxidize brominated methanes. Two distinct cultures of nonmethanotrophic methylotrophs were recovered; one of these cultures was able to utilize CH₂Br₂ as a sole carbon source, and the other was able to utilize CH₃Br as a sole carbon source.     

Optimization of the culture condition for an antitumor bacterium Serratia proteamacula 657 and identification of the active compounds

Exploration of novel active anti-tumor compounds from marine microbes for pharmaceutical applications has been a continuously hot spot in natural product research. Bacterial growth and metabolites may greatly vary under different culture conditions. In this study, the effects of different culture conditions and medium components on the growth and bioactive metabolites of Serratia proteamacula 657, an anti-tumor bacterium found in our previous study, were investigated. The results showed that lower temperature, weak acidic condition and solid fermentation favored the bacterial growth and the production of active compounds. Four components in the culture medium, NaCl, peptone, yeast extract and MgSO₄, were found important to the bacterial growth and active compounds production in medium optimization. Under the optimized condition of solid state fermentation at pH 6.0–7.0, 23–25 °C, with the MgSO₄-free medium containing 10.0 g/L peptone, 1.0 g/L yeast extract and 19.45 g/L NaCl, the antitumor activity of S. proteamacula 657 and the yield of crude extracts increased about 15 times and 6 times than the sample obtained in the original liquid fermentation, respectively. The active components in the metabolites of S. proteamacula 657 were identified as a homolog of prodigiosin, a red bacterial pigment, based on the analysis of the NMR and GC–MS. The bacterium S. proteamacula 657, which is adapted to lower temperature, produced prodigiosin-like pigments with highly antitumor activity, suggesting the bacterium is a potential new source for prodigiosin production.     

Methanogenesis from Sucrose by Defined Immobilized Consortia

A bacterial consortium capable of sucrose degradation primarily to CH₄ and CO₂ was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H₂, and CO₂ (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH₄. Significant decreases in yields of CH₄ and rates of CH₄ production were observed if any component of the consortium was omitted. Approximately 70% of the CH₄ generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH₄ and rates of CH₄ production from sucrose similar to those of nonimmobilized cells. The rate of CH₄ production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4°C.     

Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10^-0, 10^-2, 10^-4 and 10^-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.