Дисертації з теми "Phytoremediation enhanced by microorganisms"

Phytoremediation is an emerging green technology for the restoration of contaminated sites with various organic and inorganic contaminants. However, phytoremediation efficiency is limited by factors such as contaminant concentration, toxicity and bioavailability, plant choice and stress tolerance, and competence of indigenous microorganisms. A number of possible solutions have been proposed to overcome these limitations. The use of tolerant plant candidates, mixed plant communities and bioaugmentation with microbes and/or plant growth promoting bacteria (PGPB) have been proposed to suppress plant growth inhibition/phytotoxicity and enhance contaminant degradation through the rhizosphere effect but there is need for more research to understand their impact. This research assessed the impact of contaminant stress (diesel fuel, PAH; phenanthrene, fluoranthene and benzo[a]pyrene, and heavy metal) on selected plant species and microbial community structure, contribution of abiotic processes and rhizoremediation to PAH dissipation, and the impact of PGPB on plant growth and PAH dissipation. These objectives were achieved through greenhouse experiments with M. sativa, F. arundinacea and L. perenne on diesel fuel- and PAH-spiked soils. Diesel-fuel treatments had a negative impact on plant biomass yields while the single and mixed PAH treatments had stimulatory and inhibitory effects on plant biomass yields relative to the control.

Phosphorus is an essential macronutrient for plants growth, but it tends to form complexes with aluminium which limits the plant intake. Hence, phosphate solubilizing microorganisms (PSMs) serve as an alternative to solubilize and mineralize insoluble phosphate complexes. Staphylococcus haemolyticus was isolated and encapsulated in alginate microbeads by using the impinging aerosol method. This study also aimed to develop a computational fluid dynamics (CFD) model that could describe the impinging aerosol application.

One of the main routes through which pharmaceuticals may enter the environment consists in the medication of livestock. In fact, in Europe the annual national sales of active substance for veterinary consumption reach hundreds of tons. After medication, up to 90% of the administered medicine dose may be excreted unaltered and, following the use of manure as fertilizer, soils and waters are contaminated. The present work focuses on the effects and eventual accumulation on woody and herbaceous plants of sulfonamides, a group of antimicrobial agents (from now on called antibiotics) frequently detected in agricultural ecosystems, whose persistence poses a serious risk to soil and water living organisms. The thesis consists of 7 chapters, presenting, in the first one, a general introduction on the antibiotic presence in the environment and its consequences on the growth and development of exposed living organisms. Subsequently, from chapter 2 to chapter 6, various experimental trials are presented, some of them carried out under laboratory conditions and others in greenhouse. More specifically, chapter 2 reports the first study performed, which deals with Salix fragilis L. plant response and the accumulation of sulfadimethoxine antibiotic, added in the nutrient solution at doses ranging from 155 to 620 mg l-1. Such a study highlights the potential of this woody species to absorb and accumulate the active molecule at the level of root apparatus. Chapter 3 retraces the experimental design of chapter 2, with the difference that Salix fragilis L. plants were exposed to environmental relevant sulfadimethoxine doses, from 0.01 up 10 mg l-1. The trial had demonstrated that no adverse effects on the growth of willow plants appeared up to 1 mg l-1 of antibiotic. Conversely, increasing levels of the antibiotic caused important alterations of the willow root architecture. Chapters 4 and 5 consider, respectively, the effects and accumulation of a different sulfonamide on Salix fragilis L. and Zea mays L. plants, grown in a soil spiked with 10 and 200 mg kg-1 of sulfadiazine. Moreover, its impact on the composition of root associated soil microbial community and on the activities of selected enzymes was analyzed. The last study, presented in chapter 6, focuses on alterations induced by about 10 mg l-1 of sulfadimethoxine and sulfamethazine on Hordeum vulgare L. root structure and function. This chapter highlights the strong effects of the antibiotics, not only on the root apparatus morphology, but also on the membrane integrity of root cells. To conclude (chapter 7), it is highlighted that Salix fragilis L. seems to better accumulate and withstand the active molecules tested than Zea mays L. and Hordeum vulgare L., while the herbaceous species are more vulnerable to this kind of pollutant exposure and, therefore, not recommended for eventual remediation purposes. Furthermore, chapter 7 notes the adverse consequences on the functional and structural diversity of the soil microbial community.
Una delle vie principali attraverso cui i farmaci possono entrare nell'ambiente consiste nell’ampio uso che se ne fa in zootecnia. Infatti, in Europa questi principi attivi sono venduti nell’ordine di centinaia di tonnellate annue per singola nazione, per il solo utilizzo in ambito veterinario. In seguito alla somministrazione, fino al 90% della dose utilizzata di farmaco può essere escreta inalterata e, in seguito all'utilizzo del letame come ammendante organico, suolo e acque possono risultare contaminate. Il presente studio si concentra sugli effetti e sull’accumulo in piante legnose ed erbacee di sulfamidici, un gruppo di agenti antimicrobici (d'ora in poi chiamati antibiotici) frequentemente rilevati negli ecosistemi agrari, la cui persistenza rappresenta un serio rischio per gli organismi viventi ad essi connessi. La tesi è articolata in 7 capitoli. Nella prima parte (capitolo 1) è descritta la situazione generale relativa alla presenza di antibiotici negli ambienti agrari e al loro impatto sulla crescita e lo sviluppo di organismi viventi ad essi esposti. Successivamente, dal capitolo 2 al capitolo 6, sono presentate varie prove sperimentali, alcune effettuate in laboratorio ed altre in serra. In particolare, il capitolo 2 si occupa della risposta di piante di Salix fragilis L. all’antibiotico sulfadimetossina, aggiunto alla soluzione nutritiva in concentrazioni da 155 a 620 mg l-1, nonché del potenziale accumulo nei tessuti vegetali. Lo studio mostra la tendenza di questa specie legnosa di assorbire e accumulare la molecola attiva a livello di apparato radicale. Il capitolo 3 ripercorre il disegno sperimentale adottato nella prova descritta nel capitolo 2, con la differenza che, in questo caso, le piante di Salix fragilis L. sono state esposte a dosi di sulfadimetossina che approssimano quelle registrate in alcuni ambientali agrari, ovvero da 0.01 a 10 mg l-1. Lo studio ha mostrato che non appaiono effetti negativi sulla crescita delle piante di salice fino alla dose di 1 mg l-1. Tuttavia, aumentando il livello del principio attivo sono state evidenziate delle importanti alterazioni sull’architettura radicale. I capitoli 4 e 5 considerano, rispettivamente, gli effetti e l'accumulo di un altro sulfamidico in piante di Salix fragilis L. e Zea mays L., coltivate in un terreno arricchito con 10 mg e 200 kg-1 di sulfadiazina e il suo impatto sulle comunità microbiche e sulle attività enzimatiche associate al suolo e alla radice delle due specie vegetali. L'ultimo studio, presentato nel capitolo 6, si concentra sugli effetti indotti da circa 10 mg l-1 di sulfadimetossina e sulfametazina sulla struttura e sulla funzionalità di radici di Hordeum vulgare L. I risultati provano che i sulfamidici causano importanti effetti sulla morfologia dell'apparato radicale e sull’integrità delle membrane delle cellule radicali. Concludendo, si è evidenziato (capitolo 7) che il Salix fragilis L. accumula e tollera meglio di Zea mays L. e Hordeum vulgare L. le molecole attive testate, mentre le specie erbacee sembrano essere più vulnerabili a questi inquinanti, di cui ne viene sconsigliato l’eventuale utilizzo nel campo del fitorimedio. Inoltre, in capitolo 7 rimarca le conseguenze negative sulla diversità funzionale e strutturale delle comunità microbiche del suolo.

Strategies for the enhanced biological degradation of pesticides were examined in this research project. In one approach, the concept of the plant—microbial rhizosphere association was investigated as a laboratory model using the herbicide 2,4-D as a test compound. In another, an enhanced degradation of the recalcitrant herbicide atrazine was shown. Here, two microbial populations each capable of rapid atrazine metabolism were studied. The metabolism of 2,4-D by bacteria associated with the root system of wheat and canola seedlings was demonstrated in this study using a hydroponic system as well as a solid medium of sand and gravel. Significant and rapid 2,4-D metabolism (near 100% within 24h) was found in all hydroponic systems where the 2,4-D degrading microorganisms, Acinetobacter baumannii pJP4 transconjugant, Alcaligenes eutrophus and Azospirillum brasilense pIP4 transconjugant were associated with the roots. The metabolism of 2,4-D by Azospirillum brasilense pJP4 transconjugant associated with wheat was less rapid than associations with the other 2,4-D degrading bacteria. There was little difference in the rates of degradation between the hydroponic system and the sand/gravel mixture. The colonisation of the roots of seedlings by microbes was studied by both fluorescence and laser scanning confocal microscopy. Colonisation was often prolific without favoured areas of attachment on the root. A pre-treatment of seedlings with a synthetic auxin which formed para-nodular structures had little effect on the nature of colonisation. Counts of colony forming units, however, established that there was an increase of an order in magnitude of cells per root system when the plants were pretreated with this synthetic auxin. An average of 5.5 x 106 viable cells of 2,4-D degrading Acinetobacter baumannii were counted on para-modulated wheat root systems. It was demonstrated that the colonisation of the rhizosphere by suitable microbes could protect canola seedlings against phytotoxic effects of the applied herbicide. Whether this bio-safening effect will be seen in solid media or in field situations with these nonleguminous plants was not investigated. Significant rates of atrazine degradation either in the laboratory or in the field have rarely been reported. Attempts were therefore made to obtain microbes capable of such metabolism. These attempts had the ultimate goal of providing microbes for application in the model plant microbial rhizosphere association. Two microbial cultures, each capable of rapid atrazine metabolism, were obtained and studies of the metabolic processes were conducted. Rhodococcus sp. NI86/21 metabolised atrazine within l44h to two N—dealkylated products, desisopropylatrazine and desethylatrazine. Mineralisation of the ethyl-14C labelled sidechain to 14CO; was demonstrated, accounting for 25% of the total applied label in the broth culture. Desisopropylatrazine was shown to be the major metabolite. Desethylatrazine was shown as a terminal metabolite in the degradation of atrazine by Rhodococcus sp. N186/21, accumulating in the broth. In other studies using it as the substrate, no firrther metabolism was found. Desisopropylatrazine was also indicated to have been a terminal metabolite as it too accumulated in the broth. The metabolism of prometryn, a thio-ester (non-chlorinated) s-triazine was also investigated in these studies. Although Rhodococcus Sp. N186/21 did not rapidly degrade prometryn under similar conditions to those used with the atrazine assays, a mono-N-dealkylated metabolite was identified by mass-spectrometry. Possibly, prometryn was toxic to the Rhodococcus at the concentration used (lOug mL'l), as only minimal growth of the bacteria in the broth was observed by increased absorbance. Such a toxic effect was likely to have inhibited the N-dealkylation metabolism of the prometryn. In another approach, soil from a pesticide sullage site on a farm in northern NSW was assayed for its atrazine metabolising ability. Although there was no initial activity, after 30 months of perfirsion of the soil with a concentrated solution of the herbicide, it had acquired the ability to rapidly mineralise atrazine. A rapid conversion of the three carbons in the s—triazine ring to C02 was demonstrated using radiolabelled atrazine. Also, the labelled carbon in the ethyl sidechain of atrazine was rapidly metabolised to CO2. The sidechain 14C label was mineralised to 14C02 at a slower rate than the carbons in the ring. It was demonstrated that there was a likelihood of the sidechain carbon being incorporated into an unextractable intermediate metabolite, which was subsequently also less susceptible to attack by the microorganisms. There were no significant metabolites of atrazine accumulated in the broth. The likely presence of hydroxyatrazine was noted in the assay using uniformly ring—labelled [14C] atrazine. Hydroxyatrazine was also identified in the assay with ethyl-sidechain labelled [14C] atrazine, however, the amount detected was less. The presence of atrazine at 25pg mL-l inhibited nitrification reactions in the soil, however, at a saturating concentration of SOug mL-l there was some ammonia oxidation noted. Attempts to isolate single bacterial colonies capable of the metabolism of atrazine were unsuccessful. Although there was insufficient time to utilise these microbial cultures in the plant microbial rhizosphere associations, studies on the metabolism of atrazine have sufficiently characterised the nature of biodegradation to suggest that plant-microbial associations can be confidently tested in fiiture experiments.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.

Thesis (Ph. D.)--University of Georgia, 2002.
Directed by Bruce Lee Haines. Includes an article published in Journal of soil contamination, and articles submitted to Plant physiology, and Environmental toxicology and chemistry. Includes bibliographical references.

Although the pesticide DDT has been banned from use in Canada for more than three decades, DDT still persists in Canadian farmlands at detectable levels. Much effort, such as incineration, thermal desorption, and bioremediation, has been used to remediate DDT contaminated soils, but so far it is either too expensive or impractically slow. In this study, a three-year period of field trials was performed to investigate phytoremediation of DDT contaminated soil. In the field trials, millet, fall rye, sugar beet, potato, and pumpkin, treated with plant growth-promoting rhizobacteria (PGPR) were planted on two sites. As well, untreated plants were planted as a control. Plant growth, and 4,4’-DDT plus 4,4’-DDE concentrations in plant tissues and soil were monitored regularly. Comparing the plant growth between PGPR treated and untreated, PGPR significantly promoted the plant growth. On site 1, the root length and root weight of fall rye treated with PGPR were 16% and 44% greater, respectively, compared to the untreated plants. The root and shoot dry weights of millet treated with PGPR were 38% and 47% greater than those untreated plants. Root dry weight of sugar beet treated with PGPR was increased by 74% compared to untreated sugar beet. A significant effect of growth promotion was also observed in pumpkin and potato treated with PGPR. Following plant growth, DDT detection in plants was performed. 4,4’-DDT and 4,4’-DDE were found in plant tissues of fall rye, millet, sugar beet, and pumpkin. The concentrations of 4,4’-DDT and 4,4’-DDE in fall rye roots were 0.61 and 0.59 μg/g, respectively. In pumpkin tissues at harvest, 4,4’-DDT and 4,4’-DDE concentrations were 0.67 and 1.64 μg/g in roots, 1.06 and 2.05 μg/g in the lower stems, and 0.2 and 0.32 μg/g in the upper stems. The data indicated that it is feasible to phytoremediate DDT from contaminated soil. In addition, 4,4’-DDT concentrations in soils with different plant species were determined. In millet plot on site 1, 4,4’-DDT concentration in rhizosphere soil dropped by 41% in 2006 compared to 4,4’-DDT concentration at t0. In sugar beet plot on site 1, 28% of 4,4’-DDT dropped in rhizosphere soil in 2007. In pumpkin plot on site 1, 4,4’-DDT in rhizosphere soil was decreased by 22% in 2007. The results show that 4,4’-DDT concentration in rhizosphere soil was significantly lower than the initial level of DDT. Based on the data of 4,4’-DDT in soils and plant tissues, a mass balance was constructed and calculated. The preliminary mass balance shows that the total amount that DDT decreased in rhizopshere soil approximately equals to the total amount of DDT accumulated in plant tissues. This indicates that phytoextraction is the mechanism of DDT phytoremediation. In addition, PGPR promoted plant growth and then enhanced the phytoremediation efficiency of DDT. Therefore, the research indicates that PGPR assisted phytoremediation has a great potential for remediation of DDT and other chlorinated aromatics from impacted soil.

Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

博士
國立臺灣大學
農業化學研究所
92
Heavy metals-contaminated soils can be remediated by phytoremediation techniques. Phytoextraction accumulated toxic metals from contaminated soil into the aboveground tissue of higher plants, which were then harvested and incinerated. Some synthetic chelating agents were applied to metal-contaminated soil to increase the mobility and bioavailability of the metal in the contaminated soils and also to increase the amount of heavy metals accumulated in the upper parts of plants. Rainbow pink (Dianthus Chinensis), Vetiver grass (Vetiver zizanioides), and Indian mustard (Brassica juncea) were used in this study to test the remediation of the Cd, Zn, and Pb-contaminated soil. The objectives of this study are to assess the effect of applying EDTA on the phytoremediation of metals-contaminated soils and to assess the interactions among three metals in multiple metals-contaminated soils. Different applying methods with same amounts of EDTA are also used in this study to assess their effect on the metal concentration in the shoot of plant and on reducing the potential risk of groundwater contamination. Rainbow pink accumulated about 80 mg Cd/kg, 3700 mg Zn/kg, and 220 mg Pb/kg when it was grown in the Cd, Zn, and Pb-contaminated soil for 50 days. This plant can be used for phytoextraction of multiple metals-contaminated soils. Vetiver grass can grow well in the same concentrations of heavy metals-contaminated soil, and the growth was not affected by the toxicity of heavy metals. The concentrations of Cd and Zn in the shoots of vetiver grass were 40.7±8.28 and 1,399±132 mg/kg, respectively, and no Pb was detected. Because of the toxicity and high concentrations of multiple metals occurred in the soils, some damages were found in the growth stage of Indian mustard. The concentrations of Cd, Zn, and Pb in soil solution were significantly increased after applying 5 or 10 mmol EDTA/kg (p< 0.05). The concentrations of Cd and Pb in shoot of rainbow pink were also significantly increased after EDTA treatments (p< 0.05), but it was not significantly increased for Zn. For biological uptake of metals in contaminated soil, the EDTA treatments only significantly increased the total uptake of Pb in the shoot of rainbow pink compared with the control treatment (p< 0.001), but it was not significantly increased for Cd and Zn uptake by rainbow pink. This indicated that the EDTA treatments could be evaluated as more efficient amendment method to remove the Pb from the contaminated soil. The results indicated that the concentrations of Cd, Zn, and Pb in the soil solution of vetiver grass were also significantly increased after applying EDTA treatments (p< 0.05), especially for applying 10 mmol EDTA/kg. Even the concentrations of the three metals in soil solution changed drastically, but the concentrations of Cd and Zn in the shoot of vetiver grass only varied from 20 to 30 mg Cd/kg and from 390 to 520 mg Zn/kg, respectively. The growth of vetiver grass was not affected by the toxicity of seriously contaminated metals. The applying of different concentrations of EDTA solution only slightly decreased the biomass of vetiver grass and slightly decreased the total removal of heavy metals from the contaminated soils. Applying 2 or 5 mmol EDTA/kg significantly increased the Cd, Zn, Pb, Fe, and Mn concentration in the soil solution of single- or multiple metals-contaminated soils (p< 0.05), but it had no significantly change on the concentration of Ca and Mg. Deionized water extractable metal concentrations are also significantly increased after applying EDTA (p< 0.05). Because of the strong extraction capacity of both 0.005M DTPA (pH 5.3) and 0.05M EDTA (pH 7.0), there was no significant increase on the metal concentration of two extractions methods after applying EDTA. There was no effect of single or multiple-dose application of 4 mmol EDTA/kg on biomass and total removal of heavy metals in shoots of rainbow pink. But the multiple-dose applying EDTA decreased the Cd, Zn, and Pb concentration in soil solution or extracted solution with deionized water, and thus reduced the risk of groundwater contamination. There were some interactions among Cd, Zn, and Pb in the multiple metals- contaminated soils. The result of metals concentration and total removal in the shoots of rainbow pink showed that, without applying EDTA, adding Zn or Pb had enhancement effect on the uptake of Cd in the shoot of rainbow pink. The addition of Cd had inhibition effect on the uptake of Zn by rainbow pink. After applying EDTA, some interactions were found, and the addition of two concentrations of EDTA had greatest effect on the uptake of Pb by rainbow pink compared with the other elements. In this study, planting rainbow pink in the Cd, Zn, and Pb-contaminated soil for 50 days without adding EDTA was the most economic and efficient method to remove Cd and Zn from contaminated soil compared with other treatments. The rainbow pink can accumulate high concentration of Cd and Zn in the shoots and remove the maximum amounts of these two elements (0.26 mg Cd/plant and 11.7 mg Zn/plant), and also had less risk on the pollution of the groundwater when comparing with other treatments. The addition of EDTA significantly increased the concentration and total removal of Pb in the shoots of rainbow pink, thus to reduce the remediation time. However, the application of EDTA was potential to pollute the groundwater. The result also indicated that 5 mmol EDTA/kg was recommended because the soil used in this study is a silty clay soil.

Dissertação de mestrado em Biotecnologia
Microbial enhanced oil recovery (MEOR) is a tertiary oil recovery technique that uses microorganisms and their metabolites to recover the oil entrapped in the reservoirs. One of the main drawbacks related with the application of in situ MEOR is the shortage of microorganisms capable of growing and producing the desired bioproducts at the harsh conditions of the oil reservoir. Thus, the aim of this Thesis was the design and construction of microorganisms with the ability of producing biopolymers at high temperatures and oxygen-limiting conditions through protoplasts fusion. For this purpose, a biopolymer-producing strain (Rhizobium viscosum CECT 908) and a Bacillus subtilis strain isolated from an oil reservoir were used as parental strains. Several parameters were optimized to increase the protoplasts formation and regeneration frequencies for each parental strain. The most favourable conditions for protoplasts formation were an incubation with EDTA (2.9 g/L) for 60 min at 30°C followed by a treatment with 2 g/L of lysozyme for 1 h at 37°C for R. viscosum CECT 908, and incubation with 3 g/L of lysozyme for 1 h at 37°C without EDTA treatment for B. subtilis PX573. Other parameters were also evaluated to improve the protoplasts regeneration and fusion, including the use of different osmotic stabilizers and regeneration media, and the incubation time with the fusogenic agent. However, after protoplasts fusion, it was not possible to obtain recombinant strains combining the desired properties of the parental strains. In addition, four Paenibacillus sp. strains were identified as new and promising biopolymer producers. These isolates produced up to 30 g/L of biopolymer in the optimized culture medium, achieving apparent viscosity values as high as 54000 mPa s (80 times higher when compared with R. viscosum CECT 908). In oil recovery assays performed in sand-pack columns using a heavy crude oil (η40 ºC≈545 mPa s), these biopolymers allowed a recovery of 41.9 ± 0.7% of the entrapped oil, thus being promising candidates for application in MEOR. The effect of the combination of biosurfactants and biopolymers in oil recovery was also studied using the same crude oil. The results obtained demonstrated that the combination of biopolymer (R. viscosum) and surfactin (B. subtilis) resulted in similar oil recoveries (47.2 ± 0.7%) when compared with the biopolymer alone (46.2 ± 3.8%), whereas surfactin alone was unable of recovering this type of oils.
A recuperação avançada de petróleo com microrganismos (MEOR) é uma técnica de recuperação terciária que usa microrganismos e os seus metabolitos para recuperar o óleo que ficou preso nos reservatórios. Uma das principais desvantagens na aplicação de processos de MEOR in situ é a escassez de microrganismos capazes de crescer e produzir os bioprodutos desejados nas condições adversas do reservatório petrolífero. Assim, o objetivo desta tese foi a construção de microrganismos capazes de produzir biopolímeros quando expostos a altas temperaturas e em condições limitantes de oxigénio através de fusão de protoplastos. Nesse sentido, uma estirpe produtora de biopolímero (Rhizobium viscosum CECT 908) e a estirpe Bacillus subtilis, isolada a partir de um reservatório petrolífero, foram usadas como estirpes parentais. Diversos parâmetros foram otimizados de modo a aumentar as frequências de formação e regeneração de protoplastos para cada estirpe parental. As condições mais favoráveis para a formação de protoplastos foram uma incubação com EDTA (2.9 g/L) durante 60 min a 30°C seguida de um tratamento com 2 g/L de lisozima durante 1 h a 37°C para R. viscosum CECT 908, e a incubação com 3 g/L de lisozima durante 1 h a 37°C sem tratamento com EDTA para B. subtilis PX573. Outros parâmetros foram também avaliados para melhorar a regeneração e fusão de protoplastos, incluindo o uso de diferentes estabilizadores osmóticos e meios de regeneração, e o tempo de incubação com o agente fusogénico. Contudo, após a fusão de protoplastos, não foi possível obter estirpes recombinantes contendo as propriedades desejadas das estirpes parentais. Adicionalmente, quatro estirpes de Paenibacillus sp. foram identificadas como novas e promissoras produtoras de biopolímeros. Estes isolados produziram até 30 g/L de biopolímero no meio de cultura otimizado, atingindo valores de viscosidade aparente de 54000 mPa s (80 vezes maiores quando comparado com o biopolímero de R. viscosum CECT 908). Nos ensaios de recuperação de óleo em colunas de areia usando um óleo de elevada viscosidade (η40ºC≈545 mPa s), estes biopolímeros permitiram a recuperação de 41.9 ± 0.7% do óleo retido, sendo candidatos promissores para a aplicação em MEOR. O efeito da combinação de biosurfactante e biopolímero na recuperação de óleo foi também estudado usando o mesmo óleo. Os resultados obtidos demonstraram que a combinação de biopolímero (R. viscosum) e surfactina (B. subtilis) levou a recuperações de óleo semelhantes (47.2 ± 0.7%) quando comparada com o biopolímero isolado (46.2 ± 3.8%), enquanto que a surfactina isolada foi incapaz de recuperar este tipo de óleo.

Salinity is one of the most severe environmental factors that limits global crop yield. Enhanced phytoremediation using plant growth promoting rhizobacteria (PGPR) has proven to be an effective and environmentally responsible approach to remove salt from the surface soil and reclaim salt-impacted soil for crop production. PGPR enhanced phytoremediation systems (PEPS) were applied to two research sites, Cannington Manor North (CMN) and Cannington Manor South (CMS) in southern Saskatchewan. The sites were impacted by brine leakage during upstream oil and gas production. A salt mass balance study was performed based on data collected from these two sites. Both sites were planted in June. Soil samples were taken in June 2009 (beginning of the season), August (midseason) and October (end of the season). Soil salinity changes throughout the season were monitored by measuring soil electrical conductivity (EC). The average surface soil ECe decreased from 3.7 dS/m to 3.1 dS/m at CMN, and from 10.2 dS/m to 9.2 dS/m at CMS in 2009 season. Plant samples that were collected in August and October were analyzed for sodium and chloride concentrations. These values were then converted into predicted ECe changes for the soil to compare with the actual changes in soil ECe. Plant uptake of NaCl was calculated to account for 25.2% and 28.1% of the decrease in surface soil ECe at CMN and CMS, respectively. However, plant samples were washed prior to salt content analysis. A considerable amount of salt could have been lost during the washing process. Several plant samples from other salt-impacted sites in Saskatchewan and Alberta were selected to examine salt loss due to tissue washing. The salt ions lost by washing were determined to be 44.4% for Na+ and 63.8% for Cl-. After the adjustment of plant NaCl uptake data by the loss due to washing, plant accumulation of NaCl accounted for 59.9% of the decrease in surface soil ECe at CMN and 56.1% at CMS. When plant uptake of K+ and Ca2+ were also taken into consideration by a simulation study, the decrease in surface soil ECe that was caused by plant uptake of salt ions accounted for 107.5% at CMN and 117.5% at CMS. This indicated that plants can have a significant role in the remediation of salt-impacted soil. The effects of PGPR (Pseudomonas spp. UW4 and Pseudomonas corrugata CMH3) treatment on selected physiological indicators, such as proline, superoxide dismutase (SOD), membrane leakage and photosynthesis, were examined on annual ryegrass (Lolium multiflorum). Plants were grown under three saline conditions: non-saline topsoil, non-saline topsoil spiked with NaCl to 10 dS/m, and high saline soil collected from a salt-impacted site diluted with non-saline topsoil to reach 10 dS/m. The shoot fresh weight of plants grown in spiked salt soil decreased by 74% and in diluted salt soil by 44%, respectively, compared to control soil. Both types of salt soil increased SOD activities by approximately 50%, proline concentrations by 20 to 25 fold, and membrane leakage levels by 1.6 to 2.8 fold. Significant impairment of photosynthetic performances, as indicated by the decreases in the chlorophyll fluorescence parameters Fv/Fm, yield and qP, and a parallel increase in qN, was also observed using Pulse Amplitude Modulation (PAM) fluorometry for plants in diluted impacted soil. PGPR moderately increased fresh weight and SOD activity. Both UW4 and CMH3 significantly increased proline concentration and lowered membrane leakage relative to untreated plants. Therefore, PGPR improve plant performance under salt stress by elevating proline levels, which can act as a quencher of destructive reactive oxygen species. PGPR treatment also restored all the chlorophyll fluorescence parameters nearly to the non-stressed level, indicating protection of photosynthetic tissues of PGPR treated plants under salt stress. Overall, PEPS was successfully applied to the salt-impacted sites. Plant uptake of salt played a major role in the decrease of surface soil ECe. PGPR’s role in enhancing plant performance under salt stress was suggested by the elevated proline concentrations, the decreased membrane leakage levels and the restored photosynthetic activity.

Soil salinity affects an estimated one billion hectares worldwide. Excess salinity inhibits plant growth, limiting crop production. This is caused by osmotic stress in saline soil, nutrient imbalance and specific ion toxicity. There have been many methods of remediation investigated, including excavation, soil washing and phytoremediation. Phytoremediation involves the growth of plants on impacted soils to degrade or sequester contaminants. The remediation of salts relies on the uptake of ions into plant biomass where the salt is sequestered and the biomass can then be harvested. This method removes the salt from the site and leaves the top soil in place, which aids in revegetation after site remediation is completed. Plant-growth promoting rhizobacteria (PGPR) improves plant growth by lowering the levels of stress ethylene within the plant, thereby increasing the biomass available to sequester ions. The objectives of this research were to investigate the efficiency of phytoremediation of salt impacted soils in field remediation sites. Previously isolated strains of PGPR (UW3, Pseudomonas putida; UW4, Pseudomonas putida; and CMH3, Pseudomonas corrugata) were used in field trials involving the planting of oats (Avena sativa), annual ryegrass (Lolium multiflorum), tall wheatgrass (Agropyron elongatum) and tall fescue (Festuca arundinacea C.V. Inferno). The salt tolerance of various switchgrass (Panicum virgatum L.) cultivars (Cave-In-Rock, Southlow, Forestburg, and common) was compared to tall wheatgrass and Inferno tall fescue to investigate the potential of switchgrass for phytoremediation. Improvement of seed germination under salt stress by H2O2 pre-treatment was investigated both as an individual treatment and in combination with CMH3 treatment. The ion uptake into plant biomass was iii compared to the change in salinity, to determine how much of the decrease in site salinity is accounted for by uptake of salt by plants. H2O2 pretreatment resulted in a 50% increase in root and shoot emergence of tall wheatgrass under 75 mM NaCl stress compared to control treatments, which matched the germination improvement observed with PGPR treatment. The combination of H2O2 and CMH3 showed a similar improvement to root emergence under stress, but had no observable effect on shoot emergence when compared to the no-H2O2-no-PGPR control. Switchgrass cultivars showed a lower germination rate than tall wheatgrass at salt levels from 0 mM to 150 mM NaCl. The measured uptake of Na+, K+, Ca2+, Mg2+ and Cl- into plant biomass during a phytoremediation field trial was able to account for approximately 70% of the observed change in salinity in 2008. In 2009 the uptake of Na+ and Cl- into Kochia scoparia, a weed species that invaded the field site after a hard frost, was able to account for 36% of the observed change in salinity.

Thesis (Ph. D.)--University of Georgia, 2006.
Directed by Valentine Nzengung. For abstracat see http://getd.galib.uga.edu/hold5yr/yifru_dawit_d_200605_phd/yifru_dawit_d_200605_phd.pdf. Includes bibliographical references.