Influence of Extracellular Polymeric Substances on the Adsorption of Cadmium onto Three Bacterial Species

In this study, we measured the effect of EPS on Cd and proton adsorption behaviors by measuring the extent of adsorption onto biomass with and without the EPS removed via a cation exchange resin. We conducted both Cd adsorption experiments and potentiometric titrations of biomass using three common bacterial species: one Gram-positive (Bacillus subtilis) and two Gram-negative (Shewanella oneidensis, Pseudomonas putida) species. The Cd adsorption experiments were conducted as a function of metal loading in order to probe whether environmentally-low metal loadings lead to different adsorption mechanisms and roles for EPS than the higher metal loadings of most previous adsorption studies. We suspended each biomass sample in a solution of dissolved Cd in 0.01?M NaClO₄ at metal loadings of 1, 2, 5, and 74?μmol/g. Surface complexation modeling (SCM) was used to determine stability constants for the important Cd-bacteria complexes, and the effect of metal loading on the resulting calculated stability constant values was determined.

In general, the measured bulk Cd adsorption behavior is unaffected by EPS removal. However, our potentiometric titration results suggest that EPS removal does alter the distribution of site types, but not the mass-normalized total site concentration within the biomass. SCM suggests that high affinity sulfhydryl sites control Cd binding under low metal loading conditions for B. subtilis and P. putida, and that sulfhydryl sites are present both on the cells and within the EPS for these species. Conversely, the SCM results suggest that Cd-sulfhydryl binding is un-important on the EPS of S. oneidensis.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Role of extracellular polymeric substance (EPS) in toxicity response of soil bacteria Bacillus sp. S3 to multiple heavy metals

Heavy metal resistant bacteria are of great interest because of their potential use in bioremediation. Understanding the survival and adaptive strategies of these bacteria under heavy metal stress is important for better utilization of these bacteria in remediation. The objective of this study was to investigate the role of bacterial extracellular polymeric substance (EPS) in detoxifying against different heavy metals in Bacillus sp. S3, a new hyper antimony-oxidizing bacterium previously isolated from contaminated mine soils. The results showed that Bacillus sp. S3 is a multi-metal resistant bacterial strain, especially to Sb(III), Cu(II) and Cr(VI). Toxic Cd(II), Cr(VI) and Cu(II) could stimulate the secretion of EPS in Bacillus sp. S3, significantly enhancing the adsorption and detoxification capacity of heavy metals. Both Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation–emission matrix (3D-EEM) analysis further confirmed that proteins were the main compounds of EPS for metal binding. In contrast, the EPS production was not induced under Sb(III) stress. Furthermore, the TEM–EDX micrograph showed that Bacillus sp. S3 strain preferentially transported the Sb(III) to the inside of the cell rather than adsorbed it on the extracellular surface, indicating intracellular detoxification rather than extracellular EPS precipitation played an important role in microbial resistance towards Sb(III). Together, our study suggests that the toxicity response of EPS to heavy metals is associated with difference in EPS properties, metal types and corresponding environmental conditions, which is likely to contribute to microbial-mediated remediation.

     

Removal of Arsenic(III) from Waste Water Using Lactobacillus acidophilus

Lactobacillus acidophilus was used for the removal of As(III) from 50–2000 ppb As(III)-containing water solution. Biosorption of As(III) by L. acidophilus was dependent on concentration (50 to 2000 ppb) and time (0 to 3 h).L. acidophilus(1 mg dry wt/ml) was able to remove 30, 60, 300, 420, 600 ppb As(III) from 50, 100, 500, 1000, and 2000 ppb of As(III)-containing water solution, respectively, within 3 h at pH 7. Moreover, by increasing the biomass of L. acidophilus(2 mg dry wt/ml) removal of As(III) was enhanced 1.66, 1.33, 1.16, 1.42, and 1.33 times, respectively. Fourier transform infrared (FTIR) and electron spectroscopy for chemical analysis (ESCA) spectrum of As(III)-loaded biomass was also investigated. An FTIR sample spectrum of L. acidophilus fresh biomass and As(III)-loaded biomass showed band stretching of fresh and As(III)-loaded biomass for O-H, 3423.43 to 3385.04 cm^?1, and for C-O, 1742.82 to 1731.14 cm^?1, and signified that –OH and –CO groups were also involved in the removal of As(III) from As(III)-containing water solution.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Yield and physicochemical properties of EPS from Halomonas sp. strain TG39 identifies a role for protein and anionic residues (sulfate and phosphate) in emulsification of n‐hexadecane

In this study, we investigated the yield and physicochemical properties of the high molecular weight extracellular polymeric substance (HMW–EPS) produced by Halomonas sp. strain TG39 when grown on different types and ratios of substrates. Glucose (1% w/v) and a peptone/yeast extract ratio of 5.1 (0.6% w/v final concentration) yielded an EPS fraction (HMW‐glucose) exhibiting the highest anionic activity (20.5) and specific emulsifying activity (EI₂₄ = 100%) compared to EPS produced by cells grown on mannitol, sucrose, malt extract or no carbon source. The HMW–EPS fractions were capable of binding ≈255–464 mg of methylene blue (MB) per gram of EPS, which represents the highest reported binding of MB by a bacterial EPS. A comparative evaluation of these properties to those of commercial hydrocolloids indicated that the combined effect of protein and anionic residues of the HMW–EPS contributed to its ability to emulsify n‐hexadecane. Liquid chromatography revealed the HMW‐glucose EPS to be a heterogeneous polymer with a polydispersity index of 1.8. This work presents evidence of a correlation between the anionic nature and protein content of bacterial EPS with its emulsifying qualities, and identifies EPS produced by strain TG39 as a high MB‐binding bacterial sorbant with potential biotechnological application. Biotechnol. Bioeng. 2009;103: 207–216. © 2008 Wiley Periodicals, Inc.     

Studies on Uranium Removal by the Extracellular Polysaccharide of a Pseudomonas aeruginosa Strain

Extracellular polysaccharide (EPS) produced by a Pseudomonas aeruginosa strain BU2 was characterized for its ability to remove uranium from aqueous solution. The EPS was acidic in nature and found as a potent biosorbent for uranium (U), showing pH dependence and fast saturating metal sorption, being maximum (985 mg U g^{? 1} EPS) at pH 5.0. The polymer showed enhanced uranium sorption capacity and affinity with increasing solution pH, suggesting a preferential sorption of monovalent uranyl hydroxide ions over the nonhydroxylated divalent species. Pseudo-first-order and pseudo-second-order kinetic models were applied to the experimental data, assuming that the external mass transfer limitations in the system can be neglected and biosorption is sorption controlled. Equilibrium metal binding showing conformity to the Freundlich model suggested a multilayer sorption involving specific binding sites with affinity distribution. The presence of two types of metal binding sites corresponding to strong and weak binding affinity was interpreted from the Scatchard model equation. Uranium sorption by EPS was unaffected or only slightly affected in the presence of several interfering cations and anions, except iron and thorium. Fourier transform infrared (FTIR) spectroscopy ascertained the strong binding of uranium with the carboxylic groups of uronic acids of bacterial EPS at pH 5.0, whereas at lower pH, amino and hydroxyl groups played a major role in metal binding.     

Biorecovery of gold using cyanobacteria and an eukaryotic alga with special reference to nanogold formation – a novel phenomenon

Pro- and eukaryotic algal genera, i.e. Lyngbya majuscula, Spirulina subsalsa (Cyanophyceae) and Rhizoclonium hieroglyphicum (Chlorophyceae), were used for bio-recovery of gold (Au) out of aqueous solution. Au (III) spiked with ¹⁹⁸Au was used for the experiment. Batch laboratory experiments indicated quick metabolic independent binding of Au to the algae followed by active accumulation and subsequent reduction. Gold accumulation by different algal genera was found in order of R. hieroglyphicum > L. majuscula > S. subsalsa (3.28, 1.93 and 1.73 mg g^-1, respectively). It was observed that the algal biomass and the media used for the experiment turned purple in colour indicating reduction of Au (III) to Au (0) at intra- and extracellular level. This was confirmed by TEM studies of L. majuscula biomass exposed in HAuCl₄ solution where <20-nm-sized gold particles were found both inside as well as on the surface of the cell. Up to 90–100% of accumulated gold was recovered from the algal biomass by using nitric acid and acidic thiourea solution.     

Construction of a Chimeric Plasmid in Bacillus subtilis

Staphylococcal plasmids pTP4 (2.7 megadaltons encoding resistance to chloramphenicol) and pTP5 (2.6 megadaltons encoding resistance to tetracycline), which replicate and express resistance in B. subtilis, were found to cut by HindIII endonuclease respectively at a single site and three sites. A chimeric plasmid pTA1245 (4.1 megadaltons) was constructed from pTP4 and pTP5 by HindIII digestion and ligation with E. coli DNA ligase. pTA1245 expresses resistances to chloramphenicol and tetracycline in B. subtilis, and pTA1245 is amplified in the presence of tetracycline. A physical map of pTA1245 was constructed.     

Role of Ni-tolerant Bacillus spp. and Althea rosea L. in the phytoremediation of Ni-contaminated soils

In our current study, four nickel-tolerant (Ni-tolerant) bacterial species viz, Bacillus thuringiensis 002, Bacillus fortis 162, Bacillus subtilis 174, and Bacillus farraginis 354, were screened using Ni-contaminated media. The screened microbes exhibited positive results for synthesis of indole acetic acid (IAA), siderophore production, and phosphate solubilization. The effects of these screened microbes on Ni mobility in the soil, root elongation, plant biomass, and Ni uptake in Althea rosea plants grown in Ni-contaminated soil (200 mg Ni kg^?1) were evaluated. Significantly higher value for water-extractable Ni (38 mg kg^?1) was observed in case of Ni-amended soils inoculated with B. subtilis 174. Similarly, B. thuringiensis 002, B. fortis 162, and B. subtilis 174 significantly enhanced growth and Ni uptake in A. rosea. The Ni uptake in the shoots and roots of B. subtilis 174-inoculated plants enhanced up to 1.7 and 1.6-fold, respectively, as compared to that in the un-inoculated control. Bacterial inoculation also significantly improved the root and shoot biomass of treated plants. The current study presents a novel approach for bacteria-assisted phytoremediation of Ni-contaminated areas.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Impact of fluorides on the removal of heavy metals from an electroplating effluent using a flocculent brewer’s yeast strain of Saccharomyces cerevisiae

Abstract

Besides several toxic heavy metals, electroplating effluents can have in solution different cations and anions, which may influence heavy metals removal by the biomass. Among them, fluorides are commonly used in the electroplating industries and thus can be found in the respective wastewaters. In the present work, the effect of the presence of fluorides in the efficiency of chromium(III), copper(II) and nickel(II) removal, from an effluent, by heat-inactivated cells of a brewing flocculent strain of Saccharomyces cerevisiae was evaluated. The presence of fluorides severely decreased (>60%) the removal of chromium(III) by yeast biomass. This effect impaired the effective treatment of the effluent according to the US Environmental Protection Agency and the Portuguese law; conversely, a higher removal of copper(II) and nickel(II) was observed. This behaviour can be understood by metal speciation. In the presence of fluorides, chromium(III) was mainly complexed, becoming unavailable for yeast accumulation; this effect decreased the efficiency of chromium(III) removal. Thus, in the presence of fluorides, less chromium(III) is associated with biomass and consequently more yeast binding sites remain available for the uptake of other metals present in solution. This fact explains the increase of copper(II) and nickel(II) removal in the presence of fluorides.     

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.     

Screening of waste biomass from Saccharomyces cerevisiae,Aspergillus oryzae and Bacillus lentus fermentations for removal of Cu,Zn and Cd by biosorption

Three by-products of fermentations containing Bacillus lentus, Aspergillus oryzae or Saccharomyces cerevisiae biomass were tested for the capacity to absorb Cu, Cd and Zn. The composition of the three biomasses was first determined and showed high contents of ashes in both B. lentus and A. oryzae biomass and high amounts of lipids in the bacterial biomass. Metal ion binding experiments were performed by contact of 0.1 g of biomass (protonated for all the metal tests and not protonated only for the Cd test) with 50 ml of solutions containing each of the metals in the concentration range from 10 to 500 mg/ml, at pH 4.5, 3.5 and 2.5. The final metal ion concentrations were determined using a plasma absorption spectrometer, and the metal removal levels for isotherm plots were determined using the Langmuir model. The results showed that B. lentus protonated biomass had the best sorption capacity for Cu and Cd, followed by protonated A. oryzae and S. cerevisiae biomass. The sorption of Zn was low for all tested biomasses, as also was the binding of all metals at acidic pH (2.5 and 3.5). A significant increase in Cd sorption was obtained using non-protonated biomass from B. lentus and A. oryzae.     

A Comparative Study of the Biodesulfurization Efficiency of Acidithiobacillus ferrooxidans LY01 Cells Domesticated with Ferrous Iron and Pyrite

The high-sulfur coal desulfurization process completed by A. ferrooxidans LY01 cells domesticated with either ferrous iron [Fe(II)] or pyrite (FeS₂) was investigated in this article. The desulfurization rate for 13 d was as high as 67.8% for the LY01 cells domesticated with pyrite but was only 45.6% for the LY01 cells domesticated with Fe(II). Bacterial adsorption experiments indicated that the bacterial adsorption quantity onto the pyrite particles was similar to the desulfurization efficiency. FTIR analysis showed that chemical composition of the two cell types was similar, but the LY01 cells domesticated with pyrite had higher levels of hydrophobic aromatic R-O groups than cells domesticated with Fe(II). The amount of extracellular polymeric substances (EPS) from the pyrite-domesticated LY01 cells was 1820 μg C/10¹⁰ cells, which was five times more than the amount of EPS in the Fe(II)-domesticated cells; the EPS readily bound Fe(III) with a maximum binding capacity of 0.21 mg Fe(III) per mg C EPS. Strains of pyrite-domesticated LY01 with a high amount of Fe(III) in their EPS possess greater oxidation activity than Fe(II)-domesticated strains with fewer Fe(III). These experiments showed the importance of the substrate-specific differences in the oxidative activity of A. ferrooxidans LY01. In addition, this study provides theoretical guidance for the future optimization of the biodesulfurization process.     

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain,Y242

A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.     

Three-dimensional structures of the central regulatory proteins of the bacterial phosphotransferase system,HPr and enzyme IIAglc

Enzyme IIA and HPr are central regulatory proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase (PTS) system. Three-dimensional structures of the glucose enzyme IIA domain (IIA^glc) and HPr of Bacillus subtilis and Escherichia coli have been studied by both X-ray crystallography and Nuclear Magnetic Resonance (NMR) Spectroscopy. Phosphorylation of HPr of B. subtilis and IIA^glc of E. coli have also been characterized by NMR spectroscopy. In addition, the binding interfaces of B. subtilis HPr and IIA^glc have been identified from backbone chemical shift changes. This paper reviews these recent advances in the understanding of the three-dimensional structures of HPr and IIA^glc and their interaction with each other. © 1993 Wiley-Liss, Inc.     

Inhibitory effects of soluble MD‐2 and soluble CD14 on bacterial growth

The effects of the soluble forms of the endotoxin receptor molecules sMD‐2 and sCD14 on bacterial growth were studied. When Escherichia coli and Bacillus subtilis were incubated at 37°C for 18 hr with either sMD‐2 or sCD14, growth of these bacteria was significantly inhibited as evaluated by viable cell counts and NADPH/NADH activity. A mutant of sCD14 (sCD14d57‐64) lacking a region essential for LPS binding did not inhibit the growth of E. coli, whereas this mutant did inhibit the growth of B. subtilis. Addition of excess PG to the bacterial culture reversed the inhibitory effect of sMD‐2 on the growth of B. subtilis, but not on the growth of E. coli. Furthermore, when evaluated by ELISA, both sMD‐2 and sCD14 bound specifically to PG. Taken together, these results indicate that sMD‐2 and sCD14 inhibit the growth of both Gram‐positive and Gram‐negative bacteria and further suggest that binding to PG and LPS is involved in the inhibitory effect of sMD‐2 on Gram‐positive bacteria and of sCD14 on Gram‐negative bacteria, respectively.