ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

Enrichment of microbial cultures able to degrade 1,3-dichloro-2-propanol: a comparison between batch and continuous methods

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg l^-1 d^-1, as compared to 74 mg l^-1 d^-1 of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.     

Anaerobic biodegradation of pentachlorophenol in mixtures containing cadmium by two physiologically distinct microbial enrichment cultures

Anaerobic biodegradation of pentachlorophenol (PCP), in mixtures containing cadmium (Cd), by sulfidogenic (SRB) and methanogenic (MET) enrichment cultures, was studied. Removal of 91–93% of PCP occurred in both SRB- and MET-enriched cultures, in the absence of Cd, within 82 days. The presence of soluble Cd initially decreased the rate of PCP removal by the enrichment cultures, but PCP removal rates improved as the Cd precipitated. GC-MS, ¹⁴C-PCP, and ¹³C-PCP studies confirmed mineralization of PCP by both enrichment cultures, as well as the incorporation of PCP carbon into specific phospholipid fatty acids (PLFAs) of the cell membranes of PCP-degrading anaerobes. This is the first report on anaerobic biodegradation of PCP by SRB- and MET-enriched cultures in the presence, with simultaneous precipitation, of the toxic heavy metal Cd, and of the incorporation of PCP carbons into specific PLFAs of the anaerobic bacterial cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 11–17. Received 22 May 2000/ Accepted in revised form 17 March 2001     

Mechanisms of cadmium resistance in anaerobic bacterial enrichments degrading pentachlorophenol

The mechanisms of heavy-metal resistance used by adapted sulfidogenic and methanogenic enrichments degrading pentachlorophenol in the presence of cadmium (Cd) were studied. The enrichment cultures adapted to and readily tolerated bioavailable Cd concentrations up to 50 ppm while degrading an equal concentration of pentachlorophenol. Both cultures removed >95% of the Cd from solution. Transmission electron micrographs revealed (i). the presence of electron-dense particles surrounding the cells in the sulfidogenic enrichments and (ii). the unusual clumping of cells and the presence of an exopolymer in the methanogenic enrichments. Energy dispersive X-ray analysis showed that the sulfidogenic enrichments removed Cd by extracellular precipitation of cadmium sulfide, while the methanogenic enrichment culture removed Cd by extracellular sequestration of Cd into the exopolymer.     

Abundance,community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments

The study reports diversity in nitrifying microbial enrichments from low (0·5–5‰) and high (18–35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0–60 ppt).     

Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by increasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.     

植物对重金属镉的耐受机制

镉离子（Cd^2＋）具有强植物毒性,抑制植物生长,甚至使植物死亡。由于长期的环境选择和适应进化,植物发展出耐受机制,可减轻或避免Cd^2＋的毒害。硫转运蛋白、硫还原相关酶类以及半胱氨酸、谷胱甘肽和植物螯合肽合成基因的表达受Cd^2＋调控。同时这些基因的过表达也能提高植物对Cd^2＋的耐性。植物抗氧化系统对Cd^2＋胁迫诱发的活性氧的清除作用,具转运Cd^2＋活性的质膜转运蛋白促进Cd^2＋经共质体途径向木质部运输、装载,而后随蒸腾流向地上部迁移,具转运Cd^2＋活性的液泡膜转运蛋白促进Cd^2＋进入液泡的隔离作用,都在植物对Cd^2＋的耐性中起作用。     

Maximizing microbial degradation of perchlorate using a genetic algorithm: Media optimization

Microbial communities are under constant influence of physical and chemical components in ecosystems. Shifts in conditions such as pH, temperature or carbon source concentration can translate into shifts in overall ecosystem functioning. These conditions can be manipulated in a laboratory setup using evolutionary computation methods such as genetic algorithms (GAs). In work described here, a GA methodology was successfully applied to define sets of environmental conditions for microbial enrichments and pure cultures to achieve maximum rates of perchlorate degradation. Over the course of 11 generations of optimization using a GA, we saw a statistically significant 16.45 and 16.76-fold increases in average perchlorate degradation rates by Dechlorosoma sp. strain KJ and Dechloromonas sp. strain Miss R, respectively. For two bacterial consortia, Pl6 and Cw3, 5.79 and 5.75-fold increases in average perchlorate degradation were noted. Comparison of zero-order kinetic rate constants for environmental conditions in GA-determined first and last generations of all bacterial cultures additionally showed marked increases.     

Comparative evaluation of different carrier-based multi-strain bacterial formulations to mitigate the salt stress in wheat

The application of liquid bacterial consortia to soil under natural conditions may fail due to various environmental constraints. In this study, the suitability and efficiency of compost, biogas slurry, crushed corn cob, and zeolite as carriers to support the survival of plant growth-promoting rhizobacteria (PGPR) and improve the performance of multi-strain bacterial consortia to mitigate the effects of salinity stress on wheat under pot conditions were evaluated. The survival of strains of Pseudomonas putida, Serratia ficaria, and Pseudomonas fluorescens labelled with gusA was evaluated for up to 90 days. Seeds coated with different carrier-based formulations of multi-strain consortia were sown in pots at three different salinity levels (1.53, 10, and 15 dS m⁻¹). Results showed that salinity stress significantly reduced wheat growth, yield, gas exchange, and ionic and biochemical parameter values, but the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing multi-strain consortium used mitigated the inhibitory effects of salinity on plant growth and yield parameters. However, carrier-based inoculation further improved the efficacy of multi-strain consortium inoculation and significantly (P < 0.05) increased the growth, yield, and physiological parameters value of wheat at all salinity levels. On the basis of the observed trends in survival and the outcomes of the pot trials, the inoculation of multi-strain consortia in compost and biogas slurry carriers resulted in more successful wheat growth under salinity stress compared to that in the rest of the treatments tested.     

Millimeter‐scale resolution of trace metal distributions in microbial mats from a hypersaline environment in Baja California,Mexico

Microbial mats from two ponds with different salinities from the saltern of Guerrero Negro (Mexico) points toward millimeter‐scale coherent variations in trace metal (Me) concentrations (Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn). Total, HCl‐leachable and pyrite‐associated Me showed a trend of increasing concentrations with increasing depth suggesting gradual addition of reactive Me probably as a result of metal sulfide precipitation at depth. The trends in Me profiles can be ascribed to the establishment and maintenance of microzones that promote geochemical processes, bacterial population distributions, and differential mass transport within the mats. Degrees of trace metal pyritization (1 ± 1% for Zn to 24 ± 7% for Cd) as well as metals associated with the pyrite fraction (<1.4–36 ± 18 nmol g^?1 for Zn and Mn, respectively) were low, as expected from a reactive Fe‐limited system like Guerrero Negro. Calculated enrichment factors showed that Ni (2.6 ± 2.1), Co (5.5 ± 4.0), Pb (9.4 ± 7.4), and Cd (57 ± 39) were, on average, enriched in the microbial mats of Guerrero Negro. Natural enrichments of Cd, Pb, and Co in sediments along the coast of Baja California and metabolical requirements of Co and Ni by the predominant cyanobacteria in the Guerrero Negro mats may explain these enrichments. Metal characteristics in microbial mats could be advantageously used as biosignatures to identify their presence in the geological record or in other planetary systems.     

一株高耐镉硫酸盐还原菌的分离及脱硫性能研究

本研究从镉污染稻田水稻根际土壤中分离、纯化出一株硫酸盐还原菌SRB1-1,并对该菌株的生理生态特征、镉和盐耐受性、16S rDNA、脱硫性能及影响因子进行了系列分析。结果表明,该菌为革兰氏阴性菌,菌体弧状,对镉离子的耐受浓度可达200 mg/L,在2%的氯化钠浓度下仍可生长。对其16S rDNA的序列分析表明该菌株属于脱硫弧菌属(Desulfovibrio)。单因子实验考察温度、pH及SO_4~(2-)浓度对该菌脱硫效率的影响,正交实验确定了该菌最佳脱硫工艺条件及影响因子顺序。结果表明最佳脱硫工艺条件为pH 7.5、温度40℃、SO_4~(2-)浓度为1 000 mg/L、培养时间56 h。     

丛枝菌根真菌促进湿地植物对污染水体中镉的吸收

丛枝菌根真菌(AMF)具有促进植物生长、增强植物耐重金属毒害及修复重金属污染生境的作用.本试验于模拟湿地系统中设0、5、10和20 mg·L^-1镉浓度,对芦苇和狼尾草分别进行接种和不接种摩西斗管囊霉(Fm)、根内根孢囊霉(Ri)处理.结果表明: 镉污染降低了AMF侵染;接种AMF提高了芦苇和狼尾草的株高、干质量、叶绿素含量、地上部与根内的氮和镉含量,增强了根系对镉的富集能力,降低了镉转移系数.在5 mg·L^-1镉处理下,接种Fm的芦苇处理的各项指标均高于其他处理,其AMF侵染率为60.6%,每毫米根长的侵入点和泡囊数分别为2.3和3.7;与不接种对照相比,接种Fm的芦苇地上部与根系干质量分别提高69.1%和75.0%,地上部与根系氮含量分别提高38.7%和27.8%,叶绿素含量和株高分别增加3.8%和11.1%.湿地系统镉浓度与植株地上部和根内的镉含量呈显著正相关.在20 mg·L^-1镉处理下,接种Fm的芦苇地上部和根内镉含量达到最大值182.4和663.3 mg·kg^-1,镉转移系数最低,为0.27,而富集系数最高,为0.55.Fm+芦苇是本试验条件下吸收富集污染水体中镉的最佳组合.     

Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: implications for long-term chromate reduction

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.     

Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall

To study the mechanisms of exogenous NO contribution to alleviate the cadmium (Cd) toxicity in rice (Oryza sativa), rice plantlets subjected to 0.2-mM CdCl₂ exposure were treated with different concentrations of sodium nitroprusside (SNP, a NO donor), and Cd toxicity was evaluated by the decreases in plant length, biomass production and chlorophyll content. The results indicated that 0.1 mM SNP alleviated Cd toxicity most obviously. Atomic absorption spectrometry and fluorescence localization showed that treatment with 0.1 mM SNP decreased Cd accumulation in both cell walls and soluble fraction of leaves, although treatment with 0.1 mM SNP increased Cd accumulation in the cell wall of rice roots obviously. Treatment with 0.1 mM SNP in nutrient solution had little effect on the transpiration rate of rice leaves, but this treatment increased pectin and hemicellulose content and decreased cellulose content significantly in the cell walls of rice roots. Based on these results, we conclude that decreased distribution of Cd in the soluble fraction of leaves and roots and increased distribution of Cd in the cell walls of roots are responsible for the NO-induced increase of Cd tolerance in rice. It seems that exogenous NO enhances Cd tolerance of rice by increasing pectin and hemicellulose content in the cell wall of roots, increasing Cd accumulation in root cell wall and decreasing Cd accumulation in soluble fraction of leaves.     

Growth and mineral element composition in two ecotypes of Thlaspi caerulescens on Cd contaminated soil

The heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy metal polluted soils (metallicolous ecotype MET) and on soils with normal heavy metal content (non-metallicolous ecotype: NMET). In order to assess the extent and structure of variation in growth, shoot accumulation of Cd, Zn and mineral element (Ca, Mg, K, Fe), a MET ecotype from Belgium and a NMET ecotype from Luxembourg were studied. Seven maternal families from two populations of each ecotype were grown on both Cd and Zn contaminated soil. Although both ecotypes presented a similar heavy metal tolerance in the experimental conditions tested, they differed in several points. The MET populations had markedly higher biomass and higher root:shoot ratio compared to NMET populations. The Zn, and at lesser extent, the Cd hyperaccumulation capacity tended to be higher in the NMET populations. The same trend was observed for the foliar concentrations of Mg, Ca and Fe with NMET populations having higher concentrations compared to MET ones. Cd and Zn concentrations were negatively correlated with the biomass of both ecotype. However, the negative correlation between the Zn and biomass was much lower in MET ecotype suggesting a tighter control of internal Zn concentration in this ecotype. Finally, although the Cd phytoextraction capacity was similar in both ecotype, a higher Zn phytoextraction capacity was detected in NMET ecotype when these plants grow on moderate Cd and Zn concentrations.     

Bar-coded pyrosequencing reveals the responses of PBDE-degrading microbial communities to electron donor amendments

Polybrominated diphenyl ethers (PBDEs) can be reductively degraded by microorganisms under anaerobic conditions. However, little is known about the effect of electron donors on microbial communities involved in PBDEs degradation. Here we employed 454 Titanium pyrosequencing to examine the phylogenetic diversity, composition, structure and dynamics of microbial communities from microcosms under the conditions of different electron donor amendments. The community structures in each of the five alternate electron donor enrichments were significantly shifted in comparison with those of the control microcosm. Commonly existing OTUs between the treatment and control consortia increased from 5 to 17 and more than 50% of OTUs increased around 13.7 to 186 times at least in one of the microcosms after 90-days enrichment. Although the microbial communities at different taxonomic levels were significantly changed by different environmental variable groups in redundancy analysis, significant correlations were observed between the microbial communities and PBDE congener profiles. The lesser-brominated PBDE congeners, tri-BDE congener (BDE-32) and hexa-BDE, were identified as the key factors shaping the microbial community structures at OTU level. Some rare populations, including the known dechlorinating bacterium, Dehalobacter, showed significant positive-correlation with the amounts of PBDE congeners in the consortia. The same results were also observed on some unclassified bacteria. These results suggest that PBDEs-degrading microbial communities can be successfully enriched, and their structures and compositions can be manipulated through adjusting the environmental parameters.     

Prospecting for ice association: characterization of freeze-thaw selected enrichment cultures from latitudinally distant soils

Freeze-thaw stress has previously been shown to alter soil community structure and function. We sought to further investigate this stress on enriched microbial consortia with the aim of identifying microbes with ice-associating adaptations that facilitate survival. Enrichments were established to obtain culturable psychrotolerant microbes from soil samples from the latitudinal extremes of the Canadian Shield plateau. The resulting consortia were subjected to consecutive freeze-thaw cycles, and survivors were putatively identified by their 16S rRNA gene sequences. Even though the northerly site was exposed to longer, colder winters and large spring-time temperature fluctuations, the selective regime similarly affected both enriched consortia. Quantitative PCR and metagenomic sequencing were used to determine the frequency of a subset of the resistant microbes in the original enrichments. The metagenomes showed 22 initial genera, only 6 survived and these were not dominant prior to selection. When survivors were assayed for ice recrystallization inhibition and ice nucleation activities, over 60% had at least one of these properties. These phenotypes were not more prevalent in the northern enrichment, indicating that regarding these adaptations, the enrichment strategy yielded seemingly functionally similar consortia from each site.     

Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.