Comparative analysis of sulfated galactans from red algae by reductive hydrolysis and mild methanolysis coupled to two different HPLC techniques

The sugar determination of the sulfated galactans, agars and carrageenans of various red algae was performed using two different techniques of depolymerisation with subsequent HPLC analysis: 1) reductive hydrolysis/ HPAEC-PAD; 2) mild methanolysis/ RPLC-DR. Both techniques were optimized to release quantitatively the composite sugars (galactose, 6-O-methyl-galactose, the labile 3,6-anhydrogalactose and 2-O-methyl-3,6-anhydrogalactose residues) and precise relative response factors of authentic 3,6-anhydrogalactose were determined. The methanolysate neutralisation step, performed subsequently to methanolysis depolymerisation, was demonstrated as a key step for the quantitative recovery of the anhydrogalactose residues. The yield of the main sugars released by the two techniques were in good agreement for the commercial agarose and iota and kappa carrageenans studied. This revised version was published online in June 2006 with corrections to the Cover Date.     

Reductive dehalogenation as a respiratory process

Anaerobic bacteria can reductively dehalogenate aliphatic and aromatic halogenated compounds in a respiratory process. Only a few of these bacteria have been isolated in pure cultures. However, long acclimation periods, substrate specificity, high dehalogenation rates, and the possibility to enrich for the dehalogenation activity by subcultivation in media containing an electron donor indicate that many of the reductive dehalogenations in the environment are catalyzed by specific bacteria. Molecular hydrogen or formate appear to be good electron donors for the enrichment of such organisms. Furthermore, systems have to be employed which supply the cultures with the halogenated compounds beyond their toxicity level. All bacteria that are presently available in pure culture and grow with a halogenated compound as electron acceptor are members of new genera. Based on experimental results with the membrane-impermeable electron mediator methyl viologen, a model of the respiration system ofDehalobacter restrictus, a tetrachloroethene-dechlorinating bacterium, is presented. Further studies of the biochemistry and energetics of respiratory-dehalogenating strains will help to understand the mechanisms involved and perhaps reveal the evolutionary origin of the dehalogenating enzyme systems.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-1,2-DCE cis-1,2-dichloroethene - PCER tetrachloroethene reductase     

Roles of Thioredoxins in the Obligate Anaerobic Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum

Thioredoxin is a small ubiquitous protein that is involved in the dithiol-disulfide exchange reaction, byway of two cysteine residues located on the molecule surface. In order to elucidate the role of thioredoxin in Chlorobaculum tepidurn, an anaerobic green sulfur bacterium that uses various inorganic sulfur compounds and H2S as electron donors under strict anaerobic conditions for growth, we applied the thioredoxin affinity chromatography method （Motohashi et al., 2001）. In this study, 37 cytoplasmic proteins were captured as thioredoxin target candidates, including proteins involved in sulfur assimilation. Furthermore, six of the candidate proteins were members of the reductive tricarboxylic acid cycle （pyruvate orthophosphate dikinase, pyruvate flavodoxin/ferredoxin oxidoreductase, ~-oxoglutarate synthase, citrate lyase, citrate synthase, malate dehydrogenase）. The redox sensitivity of three enzymes was then examined： citrate lyase, citrate synthase, and malate dehydrogenase, using their recombinant proteins. Based on the information relating to the target proteins, the significance of thioredoxin as a reductant for the metabolic pathway in the anaerobic photosynthetic bacteria is discussed.     

Induction characteristics of reductive dehalogenation in the ortho-halophenol-respiring bacterium,Anaeromyxobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C dehalogenatesortho-substituted di- and mono-halogenated phenols and couples this activity to growth. Reductive dehalogenation activity has been reported to be inducible, however,this process has not been studied extensively. In this study, theinduction of reductive dehalogenation activity by strain 2CP-C is characterized. Constitutive 2-chlorophenoldechlorination activity occurs in non-induced fumarate-grown cells, with rates averaging 0.138 mol of Cl^- h^-1 mg of protein^-1. Once induced, these cultures dechlorinate 2-chlorophenol (2-CP) at rates as high as 116 mol of Cl^-1 h^-1 mg of protein^-1. Dechlorination of 2-CP is induced by phenol,2-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol,and 2-bromophenol. Of the substrates tested, 2-bromophenol shows the highestinduction potential, yielding double the 2-chlorophenol dechlorination rate when compared to other inducing substrates. No induced dechlorination is observed at concentrations less than5 M 2-CP. When fumarate cultures were diluted 100-fold, fumarate reduction rates were reduced roughly according to the dilution factor, while dechlorination rates were similar in fumarate grown cells amended with 2-CP and cells diluted 100-fold prior to the addition of chlorophenol. This indicates that the majority of the fumarate-grown cells in late log phase were not induced when exposed to inducing substrates such as 2-CP. This observation may have ramifications on the success of bioaugmentation using halorespiring bacteria, which traditionally relies on growing culturesusing more readily utilized substrates. The rapid dechlorination rate and unique induction pattern also make strain 2CP-C a promising model organism for understanding the regulationof reductive dehalogenation at the enzymatic level.     

Enhanced selection of an anaerobic pentachlorophenol-degrading consortium

A rapid enrichment approach based on a pentachlorophenol (PCP) feeding strategy which linked the PCP loading rate to methane production was applied to an upflow anaerobic sludge bed reactor inoculated with anaerobic sludge. Due to this strategy, over a 140-day experimental period the PCP volumetric load increased from 2 to 65 mg L(R)(-1) day(-1) with a near zero effluent concentration of PCP. Dechlorination dynamics featured sequential appearance of 3,4,5-chlorophenol, 3,5-chloro- phenol, and 3-chlorophenol in the reactor effluent. Profiling of the reactor population by denaturing gradient gel electrophoresis (DGGE) revealed a correlation between the appearance of dechlorination intermediates and bands on the DGGE profile. Nucleotide sequencing of newly detected 16S rDNA fragments suggested the proliferation of Clostridium and Syntrophobacter/Syntrophomonas spp. in the reactor during PCP degradation. Published by John Wiley & Sons, Inc.     

Investigation of cell exudates active in carbon tetrachloride and chloroform degradation

Contamination of groundwater by chlorinated solvents such as carbon tetrachloride (CCl4) and chloroform (CHCl3) is a widespread problem. The cell exudates from the methanogen Methanosarcina thermophila are active in the degradation of CCl4 and CHCl3. This research was performed to characterize these exudates. Examination of the influence of pH indicated that activity was greater under alkaline conditions. Rapid CCl4 degradation occurred from 35-65 degrees C, with first-order degradation rate coefficients increasing as temperature increased. It was found that proteins were not responsible for CCl4 degradation. The active agents in the cell exudates were <10 kDa in size, with degradation activity present in both 1-10 kDa and <1 kDa size ranges. Upon purification of the <10 kDa size range of the cell exudates on a C(18) chromatography column, 17 fractions (out of 100) degraded >50% of the added CCl4 in 8 h. These 17 fractions were pooled into three samples based on their elution time from the C(18) column. One of these pooled samples contained elevated levels of cobalt, zinc, and iron, at 2, 3, and 13 times the levels measured in similarly fractionated and pooled samples of medium, respectively. The UV-visible spectrum of this pooled sample had an absorption maximum at 560-580 nm, which is similar to the absorption maxima of heme (approximately 550 and 575 nm). The two other pooled samples contained elevated levels of zinc at 11 and 22 times the concentration measured in similarly fractionated and pooled samples of medium, respectively, and also contained very low levels of nickel, cobalt, and iron. This research suggests that the cell exudates from M. thermophila contain porphorinogen-type molecules capable of dechlorination, possibly excreted corrinoids, hemes, and zinc-containing molecules.     

Prebiotic Methylation and the Evolution of Methyl Transfer Reactions in Living Cells

An hypothesis is presented for theprebiotic origin of methyl groups and the evolution ofmethyl transfer reactions in living cells. This hypothesis,described in terms of prebiotic and early biotic chemicalevolution, is based on experimental observations in our laband in those of others, and on the mechanisms of enzymaticmethyl transfer reactions that occur in living cells. Ofparticular interest is our demonstration of the reductivemethylation of ethanolamine and glycine in aqueous solutionby excess formaldehyde. These reactions, involving prebioticcompounds and conditions, are mechanistically analogous tothe de novo origin of methyl groups in modern cellsby reduction of methylene tetrahydrofolate. Furthermore,modern cellular methyl transfers from S-adenosylmethionineto amine nitrogen may involve formaldehyde as anintermediate and subsequent reductive methylation, analogousto the prebiotic chemistry observed herein.     

A Role of Bradyrhizobium elkanii and Closely Related Strains in the Degradation of Methoxychlor in Soil and Surface Water Environments

We have reported that a leguminous bacterial strain, Bradyrhizobium sp. strain 17-4, isolated from river sediment, phylogenetically very close to Bradyrhizobium elkanii, degraded methoxychlor through O-demethylation and oxidative dechlorination. In the present investigation, we found that B. elkanii (USDA94), a standard species deposited in the Culture Collection, degraded methoxychlor. Furthermore, Bradyrhizobium sp. strain 4-1, also very close to B. elkanii, isolated from Japanese paddy field soil, degraded methoxychlor. These B. elkanii and closely related strains degraded methoxychlor through almost identical metabolic pathways, and cleaved the phenyl ring and mineralized. In contrast, another representative Bradyrhizobium species, B. japonicum (USDA110), did not degrade methoxychlor at all. Based on these findings, B. elkanii and closely related strains are likely to play an important role not only in providing the readily biodegradable substrates but also in completely degrading (mineralizing) methoxychlor by themselves in the soil and surface water environment.     

Aerobic Cometabolism of Chloroform and 1,1,1-Trichloroethane by Butane-Grown Microorganisms

Aerobic cometabolism of chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA) was observed by subsurface microorganisms grown on butane. Studies performed in batch incubated microcosms were screened for CF transformation potential using the following cometabolic substrates: ammonia, methane, propane, butane, propene, octane, isoprene, and phenol. CF transformation was observed in microcosms fed ammonia, methane, propane, and butane. The butane microcosms achieved the most effective transformation. The transformation of CF and 1,1,1-TCA was strongly correlated with butane utilization and oxygen consumption. CF transformation ceased in the absence of butane or when oxygen was depleted to low concentrations in the microcosms. No transformation of carbon tetrachloride was observed. With successive additions of CF and butane to the microcosms, complete transformation of CF was achieved at solution concentrations as high as 1 mg/L. High CF concentrations appeared to inhibit butane utilization. Maximum transformation yield (T_y) of 0.01 mg CF trans-formed/mg of butane consumed, were achieved. The results indicate that a monooxygenase enzyme required for butane utilization is likely responsible for the transformation of CF. Chloride measurements demonstrated that CF was completely dechlorinated. Approximately 70% of the chloride in the transformed 1,1,1 -TCA was released into solution, indicating incomplete dechlorination of 1,1,1-TCA. The results indicate that butane is a promising cometabolic substrate for the transformation of chlorinated methanes, chlorinated ethanes, and potentially chlorinated ethenes.     

水稻土泥浆体系中多氯联苯的微生物厌氧脱氯

考察了厌氧水稻土泥浆体系中高氯代多氯联苯混合物Aroclor1260的脱氯过程,并对体系中的微生物群落结构变化进行分析.结果表明： Aroclor1260可在厌氧水稻土泥浆体系中发生脱氯,经过128 d,总消减率达到55.5%,在泥浆体系中引入驯化的脱氯富集培养体反而使脱氯效果下降,消减率为46.9%.Aroclor1260的主要脱氯过程发生在五、六、七氯联苯,其中七氯联苯脱氯过程最显著,五氯联苯作为脱氯产物有一定累积.有机物厌氧发酵产生的H₂会被脱氯过程所消耗,从而将体系中的氢分压维持在较低水平,抑制产甲烷过程而保证脱氯过程的持续进行.不同条件和培养方式驯化得到的微生物群落结构差异较大,富集培养体引入可能导致其与原体系中脱氯相关菌群竞争,从而改变体系原有菌群结构,这可能是导致其脱氯效率下降的原因.