Characterization of a Sinorhizobium Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil

A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of Sinorhizobium meliloti. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to Sinorhizobium fredii. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.     

Methanotrophic Bacteria and Facilitated Transport of Pollutants in Aquifer Material

In situ stimulation of methanotrophic bacteria has been considered as a methodology for aquifer remediation. Chlorinated aliphatic hydrocarbons such as trichloroethylene are fortuitously oxidized by the methane monooxygenase produced by methanotrophic bacteria. Experimental results are presented that indicate that both colloidal suspensions containing methanotrophic cells and the soluble extracellular polymers produced by methanotrophic cells have the potential to enhance the transport and removal of other environmental contaminants such as polynuclear aromatic hydrocarbons and transition metals in aquifer material. Three well-characterized methanotrophic bacteria were used in the experiments: Methylomonas albus BG8 (a type I methanotroph), Methylosinus trichosporium OB3b (a type II methanotroph), and Methylocystis parvus OBBP (a type II methanotroph). Isotherms were obtained for sorption of two radiolabeled pollutants, [¹⁴C] phenanthrene and ¹⁰⁹Cd, onto an aquifer sand in the presence and absence of washed cells and their extracellular polymer. Column transport experiments were performed with the washed methanotrophic cells and phenanthrene. The distribution coefficients for Cd with extracellular polymers were of the same order as that obtained with the aquifer sand, indicating that polymers from the methanotrophic bacteria could act to increase the transport of Cd in a porous medium. Polymer from BG8 significantly reduced the apparent distribution coefficient for Cd with an aquifer sand. [¹⁴C] phenanthrene also sorbed to extracellular polymer and to washed, suspended methanotrophic cells. The exopolymer of BG8 and OBBP significantly reduced the apparent distribution coefficient (K_d) for phenanthrene with aquifer sand. The distribution coefficients for phenanthrene with the methanotrophic cells were an order of magnitude greater than those previously reported for other heterotrophic bacteria. Cells of the methanotrophs also significantly reduced the apparent K_d for phenanthrene with an aquifer sand. The three strains of methanotrophs tested displayed mobility in a column of packed sand, and strain OBBP reduced the retardation coefficient of phenanthrene with an aquifer sand by 27%. These data indicate that both extracellular polymer and mobile cells of methanotrophic bacteria display a capacity to facilitate the mobility of pollutant metals and polynuclear aromatic hydrocarbons in aquifer material.     

Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti

Exopolysaccharide production by Sinorhizobium meliloti is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners. S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 microM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.     

Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.     

Identification and quantification of a messenger ribonucleic acid induced by polynuclear aromatic hydrocarbons--using a cloned human cytochrome P-450 gene

We have isolated four overlapping human genomic clones associated with the polynuclear aromatic hydrocarbon-induced form of cytochrome P-450. The form of P-450 most closely associated with polynuclear aromatic hydrocarbons induction has been defined as P1-450. These four overlapping genomic clones span a total of 31.0 X 10(3) base pairs in length with the coding sequence lying in the center of these clones. Translation in vitro of 3-methylcholanthrene-induced mRNA, selected with the human P1-450 genomic clone, detect a protein with Mr 52000, which is immunoprecipitable by the anti-(mouse P1-450) antibody. The isolated human P1-450 genomic clone hybridizes to 3-methylcholanthrene-induced mRNA from monkey liver, benzanthracene and 3-methylcholanthrene-treated human mammary tumor cells (MCF-7), but not to isosafrole-treated human cells. Upon treatment with polynuclear aromatic hydrocarbons there is a positive correlation between induced arylhydrocarbon hydroxylase (flavoprotein-linked monoxygenase) activity and the amount of mRNA that hybridizes to the isolated human genomic clone for P1-450. The size of mRNA, induced from human cells and monkey liver by polynuclear aromatic hydrocarbons, is around 3.3 X 10(3) base pairs, which is the same as the larger of two mRNA induced by polynuclear aromatic hydrocarbons in the inbred strain of mouse (C57BL/6N). Our data also showed that the isolated DNA clone can detect a mRNA size of 3.3 X 10(3) base pairs from phytohemagglutinin-activated, benzanthracene-treated human lymphocytes. Densitometer scanning indicated the presence of a 3.6-fold variation (highest-lowest) in the levels of lymphocyte P1-450 mRNA contents among six individuals studied.     

Composition and Role of Extracellular Polymers in Methanogenic Granules

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.     

Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.     

The formation and occurrence of polynuclear aromatic hydrocarbons associated with food

Polynuclear aromatic hydrocarbons are common contaminants of processed food, usually at trace levels. These hydrocarbons are products of combustion and pyrolysis, and are present in petroleum and coal, and in products derived from them. Most polynuclear aromatic hydrocarbons are not carcinogenic, but some of them are, and a few are potent inducers of skin and lung tumors in mice. Their carcinogenic properties have not been fully explored, but they seem to be less potent by ingestion or inhalation, and they are known as a group to produce cancer in humans. The most effective carcinogens among them are those with 5 or 6 fused rings, and these tend to be less prevalent in mixtures than the 3- and 4-ring hydrocarbons, most of which are not carcinogenic. Sophisticated analytical methods, using solvent extraction and chromatography have been developed to detect and measure polynuclear aromatic hydrocarbons at levels of 1 in 10(9) (1 part per billion) or less, and these have been applied to the measurement of individual compounds in foods, as well as in products of combustion and pyrolysis. Wood smoke and smoked foods contain the carcinogenic benzo[a]pyrene at levels of 1 ppb, and other hydrocarbons; liquid smoke has lower levels. Crude vegetable oils have higher concentrations, but purified 'deodorized' oils have benzo[a]pyrene levels near 1 ppb. Sausages cooked over burning logs had as much as 200 ppb benzo[a]pyrene. Charcoal-broiled steaks and ground meat had benzo[a]pyrene concentrations up to 50 micrograms/kg, while less fatty pork and chicken had lower concentrations (up to 10 micrograms/kg). It was probable that the rendered fat dripped on to the hot charcoal and pyrolyzed to form quantities of polynuclear aromatic hydrocarbons, which rose with the smoke to deposit on the meat. Therefore, oven cooking or cooking with a heat source above the meat, or segregation of the meat from the smoke resulted in food containing negligible amounts of polynuclear aromatic hydrocarbons. Modifications of cookings practices accordingly would greatly reduce exposure to this group of carcinogens.     

Carcinogen activation by human liver enzymes in the Ames mutagenicity test.

Liver post-mitochondrial supernatants derived from 10 individuals were used as the source of metabolic activation for carcinogens in the Ames quantitative mutagenicity test using Salmonella typhimurium TA 100. The liver samples were obtained from brain-dead donors and autopsy cases. The ability of human enzymes to activate aromatic amines ranged from the undetectable to highly active for 2-acetylaminofluorene. None of the samples exhibited any ability to activate benzidine. A generally low activity was observed in the capability of human enzymes to activate the polynuclear aromatic hydrocarbons, 3-methylcholanthrene and benzo(a)pyrene. Most samples were positive for activating 4-nitrobiphenyl. However, the highest mutagenic activity in the presence of human enzymes was consistently observed for aflatoxin B1 and sterigmatocystin. These results indicated that (a) human enzyme systems, like rodent systems, are more effective in inducing mutagenic activity from mycotoxins than aromatic amines and polynuclear aromatic hydrocarbons, and (b) samples derived from different individuals exhibited considerable variation in the ability to activate carcinogens belonging to a same class of compound.     

Characterization and emulsifying property of a carbohydrate polymer produced by Bacillus pumilus UW-02 isolated from waste water irrigated agricultural soil

Bacillus pumilus UW-02, an isolate from agricultural soil irrigated with waste water was found to produce a carbohydrate polymer in the form of extracellular polysaccharide (EPS) in glucose mineral salts medium (GMSM). The recovery rates of EPS by ion-exchange and gel filtration chromatography were around 63% and 90%, respectively. As evident from HPLC and FT-IR analyses, the EPS was found to be a heteropolymer consisting glucose, mannose, xylose, arabinose, and N-acetyl glucosamine as monomer units. Different oligosaccharide combinations namely hexose(4), hexose(6) pentose(1) and hexose(10) pentose(1) are obtained after partial hydrolysis of EPS using MALDI-ToF-MS. Electron micrographs portrayed the intense affinity of the EPS molecules for each other, thereby justifying its viscosifying and thickening properties. The EPS with an average molecular weight of 218 kDa and thermal stability up to 180 °C showed pseudoplastic rheology and significant emulsifying activities.     

Assessment of occupational exposure to PAHs in an Estonian coke oven plant- correlation of total external exposure to internal dose measured as 1-hydroxypyrene concentration

The exposure of cokery workers to polynuclear aromatic hydrocarbons at an Estonian oil shale processing plant was assessed by using occupational hygiene and biomonitoring measurements which were carried out twice, in midwinter and in the autumn. To assess the external dose of polynuclear aromatic hydrocarbons, pyrene and benzo[a]pyrene concentrations were measured from the breathing zone of workers during a workshift. Skin contamination with pyrene and benzo[a]pyrene was assessed by skin wipe sampling before and after the workshift. As a biomarker of overall exposure to polynuclear aromatic hydrocarbons, and as an integral of all absorption routes of pyrene, 1-hydroxypyrene concentration was measured from post shift urine samples. Of the personal air samples, 18% exceeded the Finnish threshold limit value of benzo[a]pyrene (10 μg m^-3). Mean value (two separate measurements together) for benzo[a]pyrene was 5.7 μg m^-3 and for pyrene, 8.1 μg m^-3. Based on skin wipe sample analyses, the skin contamination was also obvious. The mean value of benzo[a]pyrene in the samples collected after the shift was 1.2 ng cm^-2. Benzo[a]pyrene was not found in control samples. The mean value of urinary 1-hydroxypyrene concentration was 6.0 μmol mol^-1 creatinine for the exposed workers and 0.5 μmol mol^-1 creatinine for the controls. This study undoubtedly shows the usefulness of 1-hydroxypyrene as an indicator of internal dose of polynuclear aromatic hydrocarbons. It can be concluded that the cokery workers at the Kohtla-Järve plant are exposed to high concentrations of polynuclear aromatic compounds, and the exposure level is considerably higher during the winter measurements.     

Impact of sewage sludges on Medicago truncatula symbiotic proteome

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M. truncatula symbiotic proteome.     

Infectivity of Rhizobium meliloti as affected by extracellular polysaccharides

O livares , J., B edmar , E.J. & M artinez -M olina , E. 1984. Infectivity of Rhizobium meliloti as affected by extracellular polysaccharides. Journal of Applied Bacteriology 56 , 389–393.
The addition of crude extracellular polysaccharide (EPS) preparations from highly infective Rhizobium meliloti increased the infectivity of a poorly infective strain. The addition of crude EPS preparations from another species did not promote nodu-lation, suggesting the specificity of such an effect. Acid treatment of the crude EPS preparations resulted in total inhibition of nodulation.     

Use and improvement of microbial redox enzymes for environmental purposes

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.     

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g⁻¹ of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.     

Compositional changes during landfarming of weathered michigan crude oil‐contaminated soii

Laboratory landfarming experiments were conducted to study the bioremediation potential of weathered Michigan crude oil‐contaminated soils. It was found that landfarming was successful in removing up to 90% of the total petroleum hydrocarbons (TPH) in the soil within 22 weeks of treatment. Boiling point analyses of untreated and treated soils indicate a significant removal of TPH compounds independent of molecular weight or carbon number. Up to 85% of heavy petroleum hydrocarbons with carbon numbers above 44 were biode‐graded. In addition, approximately 93% of saturated and 79% of aromatic compounds of the TPH were biodegraded during the 22 week treatment period. The use of polyethylene sheeting as a landfarm cover does not appear to adversely affect biodegradation kinetics under laboratory conditions. Finally, equilibrium leachate concentrations for BTEX and regulated (in Michigan) polynuclear aromatics (PNAs) were below the respective detection limits for each compound. It can be concluded that landfarming of these weathered soils will be highly successful in removing petroleum hydrocarbons while not adversely impacting either ground‐water or surface water quality.     

Rhizobial exopolysaccharides: genetic control and symbiotic functions

Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral β-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear.     

石油污染土壤水改旱田后污染物组份及微生物群落结构变化

对沈抚灌区水改旱田不同年限土壤的石油污染物浓度及组分进行了分析,并采用变性梯度凝胶电泳和磷脂脂肪酸分析方法,分析了污染土壤微生物群落结构的变化.结果表明：1)石油污染土壤水改旱田后,年限越长,总多环芳烃在总石油烃中所占的比重越大,高分子量多环芳烃在总多环芳烃中所占比重也越大;2)总磷脂脂肪酸量与总石油烃呈显著正相关,与总多环芳烃相关性不显著;3)两种方法对土壤微生物群落结构的分析得出的结论一致,石油污染土壤微生物群落结构主要与其相对地理位置有关,当污染物的浓度达到一定程度时,土壤微生物群落结构会发生明显的改变.     

土壤中高环多环芳烃微生物降解的研究进展

微生物修复是去除土壤中多环芳烃(PAHs)的主要措施。本文以微生物修复PAHs污染土壤的理论基础及其难点为主线,全面综述了土壤中高环PAHs的微生物降解机理。近年来,富集分离得到的以高环PAHs为唯一碳源和能源的优势降解菌逐渐增多,其中,主要是代谢降解四环PAHs的单株降解菌,一些降解菌还能以共代谢方式利用五环PAHs。高环PAHs污染土壤修复的一个难点是其低生物可利用性,微生物通过释放生物表面活性剂、形成生物膜以及分泌胞外多糖提高高环PAHs的生物可利用性,从而加速其降解。真菌和细菌联合作用能增强污染土壤实地修复的效果。因此,通过微生物修复技术来去除土壤中PAHs具有环境友好性、经济适用性以及可持续应用性。     

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.