Contrasting the Pb (II) and Cd (II) tolerance of Enterobacter sp. via its cellular stress responses.

Successful application of microorganisms to heavy metal remediation depends on their resistance to toxic metals. This study contrasted the differences of tolerant mechanisms between Pb2+ and Cd2+ in Enterobacter sp. Microbial respiration and production of formic acid showed that Enterobacter sp. had a higher tolerant concentration of Pb (>1000 mg l-1 ) than Cd (about 200 mg l-1 ). Additionally, SEM confirmed that most of Pb and Cd nanoparticles (NPs) were adsorbed onto cell membrane. The Cd stress, even at low concentration (50 mg l-1 ), significantly enlarged the sizes of cells. The cellular size raised from 0.4 × 1.0 to 0.9 × 1.6 µm on average, inducing a platelet-like shape. In contrast, Pb cations did not stimulate such enlargement even up to 1000 mg l-1 . Moreover, Cd NPs were adsorbed homogeneously by almost all the bacterial cells under TEM. However, only a few cells work as 'hot spots' on the sorption of Pb NPs. The heterogeneous sorption might result from a 'self-sacrifice' mechanism, i.e., some cells at a special life stage contributed mostly to Pb sorption. This mechanism, together with the lower mobility of Pb cations, caused higher microbial tolerance and removal efficiency towards Pb2+ . This study sheds evident contrasts of bacterial resistance to the two most common heavy metals.

Screening and Characterization of Cold-Active ß-Galactosidase Producing Psychrotrophic Enterobacter ludwigii from the Sediments of Arctic Fjord.

Low-temperature-tolerant microorganisms and their cold-active enzymes could be an innovative and invaluable tool in various industrial applications. In the present study, bacterial isolates from the sediment samples of Kongsfjord, Norwegian Arctic, were screened for ß-galactosidase production. Among the isolates, KS25, KS85, KS60, and KS92 have shown good potential in ß-galactosidase production at 20 °C. 16SrRNA gene sequence analysis revealed the relatedness of the isolates to Enterobacter ludwigii. The optimum growth temperature of the isolate was 25 °C. The isolate exhibited good growth and enzyme production at a temperature range of 15-35 °C, pH 5-10. The isolate preferred yeast extract and lactose for the maximum growth and enzyme production at conditions of pH 7.0, temperature of 25 °C, and agitation speed of 100 rpm. The growth and enzyme production was stimulated by Mn2+ and Mg2+ and strongly inhibited by Zn2+, Ni2+, and Cu+. ß-Galactosidases with high specific activity at low temperatures are very beneficial in food industry to compensate the nutritional problem associated with lactose intolerance. The isolate exhibited a remarkable capability to utilize clarified whey, an industrial pollutant, for good biomass and enzyme yield and hence could be well employed in whey bioremediation.

Bacterial succession and degradative changes by biofilm on plastic medium for wastewater treatment.

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment.

Bioethanol production from mannitol by a newly isolated bacterium, Enterobacter sp. JMP3.

In this study a new bacterium capable of growing on brown seaweed Laminaria japonica, Enterobacter sp. JMP3 was isolated from the gut of turban shell, Batillus cornutus. In anaerobic condition, it produced high yields of ethanol (1.15 mol-EtOH mol-mannitol(-1)) as well as organic acids from mannitol, the major carbohydrate component of L. japonica. Based on carbon distribution and metabolic flux analysis, it was revealed that mannitol was more favorable than glucose for ethanol production due to their different redox states. This indicates that L. japonica is one of the promising feedstock for bioethanol production. Additionally, the mannitol dehydrogenation pathway in Enterobacter sp. JMP3 was examined and verified. Finally, an attempt was made to explore the possibility of controlling ethanol production by altering the redox potential via addition of external NADH in mannitol fermentation.

Cellulose synthesized by Enterobacter sp. FY-07 under aerobic and anaerobic conditions.

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. In static cultivation at 30 °C for 72 h under anoxic, oxygen-limited and aerated conditions, cellulose production exceeded 5 g/l, which indicated that oxygen was not essential for production of BC by Enterobacter sp. FY-07. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis showed that the microstructure of the BC was similar to that produced by aerobic bacteria such as Gluconacetobacter xylinum BCRC12335 and Acetobacter sp. V6. The crystallinity index of the BC was 63.3%. Water-holding capacity (approximately 11000%) and rehydration ratio (24.4%) were superior to those reported for BC produced by the aerobic bacteria G. xylinum BCRC12335 and Acetobacter sp. V6. These results will facilitate static submerged fermentation for the production of BC.

Mercury bioremediation by mercury accumulating Enterobacter sp. cells and its alginate immobilized application.

The effective microbial remediation of the mercury necessitates the mercury to be trapped within the cells without being recycled back to the environment. The study describes a mercury bioaccumulating strain of Enterobacter sp., which remediated mercury from the medium simultaneous to its growth. The transmission electron micrographs and electron dispersive X-ray analysis revealed the accumulation of remediated mercury as nano-size particles in the cytoplasm as well as on the cell wall. The Enterobacter sp. in the present work was able to accumulate mercury, without being engineered in its native form. The possibility of recovering the accumulated mercury from the cells is also indicated. The applicability of the alginate immobilized cells in removing mercury from synthetic and complex industrial effluent in a batch mode was amply demonstrated. The initial load of 7.3 mg l(-1) mercury in the industrial effluent was completely removed in 72 h. The cells immobilized in calcium alginate were similarly effective in the complete removal of 5 mg l(-1) HgCl(2) of mercury from the synthetic effluent in less than 72 h. The immobilized cells could be reused for multiple cycles.

Biofouling and biodeterioration in materials stored at the Historical Archive of the Museum of La Plata, Argentine and at the National Archive of the Republic of Cuba.

The aims of this paper were to study the biofouling and biodeterioration of photos and maps stored at Historical Archive of the Museum of La Plata (HAMP), Argentine, and two repositories of the National Archive of Cuba Republic (NARC) and to carry out the physiological characterization of the isolated fungi and bacteria. The role of the environmental microbiota in the biofouling formation was also studied. Microbial assemblages in the air were sampled by sedimentation technique while those on documents were sampled by swabbering. Biofilm formation and biofouling were monitored by scanning electron microscope (SEM). Large microbial assemblages were found at NARC archives with the prevalence of genera Aspergillus, Cladosporium and Penicillium, whereas at HAMP these values were lower, Penicillium was the only fungal genus detected. Most of the fungi degraded cellulose and produced pigments and acids, and all of the isolated bacteria had proteolytic and/or cellulolytic activity. In all cases, a higher concentration of viable bacteria than of fungi was isolated from documents. These results correlated with bacterial values detected in air at NARC repositories. However, this correlation cannot be observed at HAMP where Aspergillus, Penicillium and Talaromyces helicus (teleomorph of Penicillium) were isolated. It is the first time that the last genus is reported in documents.

Mercury bioaccumulation and simultaneous nanoparticle synthesis by Enterobacter sp. cells.

A mercury resistant strain of Enterobacter sp. is reported. The strain exhibited a novel property of mercury bioaccumulation with simultaneous synthesis of mercury nanoparticles. The culture conditions viz. pH 8.0 and lower concentration of mercury promotes synthesis of uniform sized 2-5 nm, spherical and monodispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanoparticles is unable to vaporize back into the environment thus, overcoming the major drawback of mercury remediation process. The mercury nanoparticles were recoverable. The nanoparticles have been characterized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder X-ray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from environmental effluent and developing a green process for nanoparticles biosynthesis.

Morphological variation of Enterobacter sp. BL-2 in acetate-mediated pH environment for excretive production of cationic microbial polyglucosamine biopolymer.

Enterobacter sp. BL-2 excretively produced unique cationic polyglucosamine biopolymer PGB-1 comprised of more than 95% D-glucosamine in an acetate-mediated culture condition. The excretion of the biopolymer PGB- was closely associated with the cellular morphology Enterobacter sp. BL-2, a feature highly dependable on the pH of the medium. The initially formed uneven and irregular surface cells were aggregated into the cell-biopolymer network structure connected by the adhesion modules of the cell-bound biopolymer. The excretive production of the biopolymer PGB-1 coincided with the disruption of the cell-biopolymer network, most actively at the medium pH of 8.0.

[Isolation and characterization of H2-producing strains Enterobacter sp. and Clostridium sp].

Two hydrogen-producing bacterial strains were newly isolated and identified as Enterobacter sp. Z-16 and Clostridium sp. C-32 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen production of strain Z-16 were achieved as: initial pH7.0, temperature 35 degrees C , sucrose as the favorite substrate. In comparison, The optimum condition for hydrogen production of strain C-32 were obtained as: initial pH8.0, temperature 35 degrees C , maltose as the favorite substrate . Under batch fermentative hydrogen production conditions, the maximal hydrogen conversion rate for strain Z-16 and strain C-32 were 2.68 mol H2/mol sucrose and 2.71mol H2/mol maltose, respectively. Using glucose as substrate, the hydrogen conversion rate of strain Z-16 and strain C-32 were 2.35 and 2.48 mol H2/mol glucose, respectively. This research suggest a good application potential of strain Z-16 and C-32 in the future biological hydrogen production.

Enhanced adsorption of zinc is associated with aging and lysis of bacterial cells in batch incubations.

Bacteria can immobilize significant quantities of trace metals through surface complexation reactions. However, bacterial cell lysis is an integral part of the development process, and the extent to which this process affects adsorbed metals has not been properly investigated. In order to evaluate the effects of cell lysis on metal fixation, bacterial suspensions containing approximately 10 ppm Zn in 0.01 M NaNO(3) were monitored over an one-month period for adsorbed Zn, pH, cell concentration, dissolved organic carbon, NH(3) and dissolved amino acids. Cell concentration decreased with time, in parallel with an increase in dissolved organic carbon. Zn adsorption decreased with time for suspensions with near-neutral (5.5-7.0) initial pH values, consistent with the reduction in cell concentration and/or formation of metal-ligand complexes in solution, with lysis products acting as ligands. However, Zn adsorption increased with time for suspensions with initially low pH (

Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses.

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is able to grow in toxic concentrations of n-butanol up to 1.5 % (volume in volume) as the sole carbon and energy source. Morphology changes in the cells growing on increasing concentrations of n-butanol were observed. The size of the bacteria decreased with increasing concentrations of n-butanol, also leading to an enhanced ratio between the surface and volume of the cells. This is in complete contradiction to the reaction of glucose-grown cells to which n-butanol had been added as a toxin. Similar differences were found in typical adaptive responses to toxic organic compounds, namely changes in fatty acid composition of membrane lipids and the activity of catalase. In both cases, reactions depending on the n-butanol concentrations could be observed when the toxin was added to glucose-grown cells, whereas no reaction was observable when the cells were growing in n-butanol as the sole carbon and energy source. This is another proof for the observation that there are certain differences between the adaptive strategies of cells when adapting to high concentrations of a growth substrate and those when adapting to a toxin added to growing cells.

Biosorption of lead, copper and cadmium by an indigenous isolate Enterobacter sp. J1 possessing high heavy-metal resistance.

This study was undertaken to investigate biosorption kinetics and equilibria of lead (Pb), copper (Cu) and cadmium (Cd) ions using the biomass of Enterobacter sp. J1 isolated from a local industry wastewater treatment plant. Efficiency of metal ion recovery from metal-loaded biomass to regenerate the biosorbent was also determined. The results show that Enterobacter sp. J1 was able to uptake over 50mg of Pb per gram of dry cell, while having equilibrium adsorption capacities of 32.5 and 46.2mg/g dry cell for Cu and Cd, respectively. In general, Langmuir and Freundlich models were able to describe biosorption isotherm fairly well, except that prediction of Pb adsorption was relatively poor with Langmuir model, suggesting a different mechanism for Pb biosorption. Adjusting the pH value to 3.0 led to nearly complete desorption of Cd from metal-loaded biomass, while over 90% recovery of Pb and Cu ions was obtained at pH

Novel method for measurement of outer membrane permeability to new beta-lactams in intact Enterobacter cloacae cells.

The ability of five new beta-lactams to permeate the outer membrane of intact Enterobacter cloacae beta-lactamase-overproducing cells was measured by using a high-pressure liquid chromatography (HPLC)-based technique that avoided certain possible artifacts of the traditional methods. Low concentrations of antibiotics were mixed with bacterial suspensions, and at different times, the cells were removed from the medium by filtration. Residual beta-lactam concentrations in the medium were then assessed by HPLC and UV detection. The assay was performed under conditions in which no beta-lactamase activity was detected in the filtrate and the number of viable cells remained constant during the experiment. Outer membrane permeability was assessed with the Zimmermann-Rosselet equation, in which outer membrane permeability was rate limiting for hydrolysis of the beta-lactam by periplasmic beta-lactamase. Thus, the rate of disappearance of beta-lactam was equal to the rate of outer membrane permeation. Preincubation of bacterial suspensions with 300 micrograms of cloxacillin per ml inhibited the hydrolysis of beta-lactams by intact cells, demonstrating that beta-lactam hydrolysis by periplasmic beta-lactamase was essential in order to allow measurement of outer membrane permeability by this method. Permeability coefficients (P) were calculated from the Zimmermann-Rosselet equation and were independent of the external concentration of antibiotic over a 100-fold concentration range. Cefepime and cefpirome exhibited rates of outer membrane permeation 5- to 20-fold higher than those of carumonam, ceftriaxone, and cefotaxime. Thus, the presence of a positive charge in the 3-lateral chain increased the permeation ability of beta-lactam molecules considerably.

Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S.

Large-scale purification of the chromosomal beta-lactamase from Enterobacter cloacae P99.

Homogeneous beta-lactamase (beta-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the beta-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.

A morphological study of the action of equine anti-lipopolysaccharide plasma on gram-negative bacteria.

Three strains of gram-negative bacteria--one each of Escherichia coli, Klebsiella pneumoniae and Enterobacter sp.--were treated with anti-lipopolysaccharide hyperimmune equine plasma (anti-LPS) or non-immune control plasma and examined by scanning electronmicroscopy. Within a few minutes of treatment with anti-LPS, bacteria were agglutinated. Evidence of cell membrane destruction was observed shortly thereafter and total cell disintegration and disruption occurred within 1-2 h. In contrast, non-immune plasma had no effect on cell morphology. This confirms the findings in previous microbiological studies that specific antibodies in anti-LPS bind to lipopolysaccharide (LPS endotoxin), and thereby initiate the destruction of gram-negative bacteria.

Attachment as a factor in the protection of Enterobacter cloacae from chlorination.

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.

Use of tannic acid for the ultrastructural visualization of periplasm in gram-negative bacteria.

Tannic acid mordanting reveals the periplasm, the area between the outer membrane and the inner membrane of gram-negative bacteria, Rhizobium sp., Escherichia coli and Enterobacter aerogenes, as an electron-dense layer continuous with the inner leaflet of the outer membrane. The method involves 18 hr of tannic acid treatment after fixation in aldehyde prior to osmium tetroxide postfixation, followed by conventional electron microscopy.

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model.

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.