This study analyzed changes in the microbial community at different stages of an air sparging bioremediation treatment of soil highly contaminated with jet fuel and BTEX compounds. Microbial community composition and biomass were monitored using phospholipid fatty acid profiling and 16S rRNA gene analysis. Initially, the community was dominated by Pseudomonads under high contamination. As treatment progressed and contaminant levels decreased, biomass and diversity increased. The community shifted to a more complex composition with diverse bacterial taxa, indicating selection pressure from contaminants was reduced as bioremediation succeeded.
Heavy metals, particularly silver and mercury, have a variety of applications in controlling microbial population. Ps. aeruginosa is a high intrinsic resistant to antibiotics and heavy metals including Copper Sulfate, Silver Sulfate, Mercury chloride, Lead nitrate, Zinc sulfate, Cadmium sulfate, and Nickel sulfate.
2005 enriqueta arias et al soil health a new challenge for microbiologist and...Germán Tortosa
The document discusses soil health and its assessment. Soil health refers to the biological, chemical, and physical features essential for sustainable agriculture with minimal environmental impact. It cannot be directly measured but can be inferred by measuring specific properties like organic matter content and observing soil status. There is interest in studying soil microorganisms and their relationship to soil structure and function. Determining soil health indicators that can evaluate current status helps develop sustainable agriculture systems. Modern techniques now enable rapid measurement of soil microbial communities and analysis of microbial diversity and activity.
Soil samples from Basra, Iraq were analyzed to assess levels of lead and cadmium contamination. Heavy metal concentrations varied between districts, with the highest levels of both metals found in Um-Qasr. Bacteria were isolated from the soils, including Deinococcus radiodurans, Shewanella oneidensis, and Bacillus thuringiensis. These bacteria exhibited varying levels of tolerance to lead and cadmium, and were able to bioaccumulate and biosorb the metals from solutions. Characterization studies showed the bacteria incorporated metals into their cells and cell walls through various functional groups, indicating their potential for bioremediating contaminated soils and water.
Assessment of remediation Potentials of maize (Zea mays) on sites co-contamin...IJAEMSJORNAL
Phytoremediation is a promising technology for the remediation of sites co-contaminated with inorganic and organic pollutants. A pot experiment was conducted to evaluate the remediation potential of Z.mays in soil co- contaminated with Pb and antracene. Pristine sandy loam soils were polluted with Pb chloride salt and antracene at three different levels (50mg/kg of Pb, 100mg/kg of Pb, and 100mg/kg of Pb+100mg/kg of antracene) and laid out in completely randomized design with 3 replicates. Shoot dry matter weight was significantly reduced (p≤0.05) when compared with control treatments by 40% when exposed to100mg kg-1 of Pb. There was a 48% inhibition of shoot dry matter of Z.mays relative to control treatments when 100 mg Pb kg-1 was mixed with 100 mgkg-1 antracene. Root and shoot metal concentration in Zea mays increased with increasing concentration of Pb. The average Translocation Factor (TF < 1 (0.69) obtained suggests that Zea mays predominantly retains Pb in the root portion of the plant. There was a 5% increase in shoot Pb concentration when soil was contaminated with Pb and antracene. The extractable antracene decreased significantly (p≤0.05) in soil planted with Z.mays as well as in pots without maize plant. This accounted for 65 and 72% of antracene dissipation in planted soil and 40-46% dissipation in unplanted soil. This result suggested that Zeamays is a promising candidate for uptake Pb and dissipation of antracene in co-contaminated soils.
mpact of Some Ecological Factors on the Occurrence and Distribution of Mitosp...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
ABSTRACT- Laccase is multicopper oxidases that are widely distributed among plants, insects, fungi and bacteria. Pollution increased with the
time day by day, laccase is an oxido-reductase which plays a significant role in remediation. These enzyme catalyze and one-electron oxidation of a
wide variety of organic and inorganic substrate including mono-, di-, and poly-phenols, amino-phenols, metho-oxyphenols, aromatic amines, and
ascorbate, with the concomitant four electron reduction of oxygen to water. Present study on their use in several industrial application, includes dye
decolorization, detoxification of environmental pollutants and revalorization of waste and waste water etc. this review helps to understand the properties
of these improvement enzymes for efficient utilization for its biotechnological and environmental applications. Now we provide a brief discussion
of this interesting group of enzymes, increase knowledge of which will promote laccase based industrial process in future.
Keywords: Laccase, Biodegradation, Bioremediation and Dye decolorization
Heavy metals, particularly silver and mercury, have a variety of applications in controlling microbial population. Ps. aeruginosa is a high intrinsic resistant to antibiotics and heavy metals including Copper Sulfate, Silver Sulfate, Mercury chloride, Lead nitrate, Zinc sulfate, Cadmium sulfate, and Nickel sulfate.
2005 enriqueta arias et al soil health a new challenge for microbiologist and...Germán Tortosa
The document discusses soil health and its assessment. Soil health refers to the biological, chemical, and physical features essential for sustainable agriculture with minimal environmental impact. It cannot be directly measured but can be inferred by measuring specific properties like organic matter content and observing soil status. There is interest in studying soil microorganisms and their relationship to soil structure and function. Determining soil health indicators that can evaluate current status helps develop sustainable agriculture systems. Modern techniques now enable rapid measurement of soil microbial communities and analysis of microbial diversity and activity.
Soil samples from Basra, Iraq were analyzed to assess levels of lead and cadmium contamination. Heavy metal concentrations varied between districts, with the highest levels of both metals found in Um-Qasr. Bacteria were isolated from the soils, including Deinococcus radiodurans, Shewanella oneidensis, and Bacillus thuringiensis. These bacteria exhibited varying levels of tolerance to lead and cadmium, and were able to bioaccumulate and biosorb the metals from solutions. Characterization studies showed the bacteria incorporated metals into their cells and cell walls through various functional groups, indicating their potential for bioremediating contaminated soils and water.
Assessment of remediation Potentials of maize (Zea mays) on sites co-contamin...IJAEMSJORNAL
Phytoremediation is a promising technology for the remediation of sites co-contaminated with inorganic and organic pollutants. A pot experiment was conducted to evaluate the remediation potential of Z.mays in soil co- contaminated with Pb and antracene. Pristine sandy loam soils were polluted with Pb chloride salt and antracene at three different levels (50mg/kg of Pb, 100mg/kg of Pb, and 100mg/kg of Pb+100mg/kg of antracene) and laid out in completely randomized design with 3 replicates. Shoot dry matter weight was significantly reduced (p≤0.05) when compared with control treatments by 40% when exposed to100mg kg-1 of Pb. There was a 48% inhibition of shoot dry matter of Z.mays relative to control treatments when 100 mg Pb kg-1 was mixed with 100 mgkg-1 antracene. Root and shoot metal concentration in Zea mays increased with increasing concentration of Pb. The average Translocation Factor (TF < 1 (0.69) obtained suggests that Zea mays predominantly retains Pb in the root portion of the plant. There was a 5% increase in shoot Pb concentration when soil was contaminated with Pb and antracene. The extractable antracene decreased significantly (p≤0.05) in soil planted with Z.mays as well as in pots without maize plant. This accounted for 65 and 72% of antracene dissipation in planted soil and 40-46% dissipation in unplanted soil. This result suggested that Zeamays is a promising candidate for uptake Pb and dissipation of antracene in co-contaminated soils.
mpact of Some Ecological Factors on the Occurrence and Distribution of Mitosp...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
ABSTRACT- Laccase is multicopper oxidases that are widely distributed among plants, insects, fungi and bacteria. Pollution increased with the
time day by day, laccase is an oxido-reductase which plays a significant role in remediation. These enzyme catalyze and one-electron oxidation of a
wide variety of organic and inorganic substrate including mono-, di-, and poly-phenols, amino-phenols, metho-oxyphenols, aromatic amines, and
ascorbate, with the concomitant four electron reduction of oxygen to water. Present study on their use in several industrial application, includes dye
decolorization, detoxification of environmental pollutants and revalorization of waste and waste water etc. this review helps to understand the properties
of these improvement enzymes for efficient utilization for its biotechnological and environmental applications. Now we provide a brief discussion
of this interesting group of enzymes, increase knowledge of which will promote laccase based industrial process in future.
Keywords: Laccase, Biodegradation, Bioremediation and Dye decolorization
This document describes Demba Jammeh's research project exploring bacterial biodiversity in acid mine contaminated sediments in Adventdalen Landfill, Svalbard, Norway. The project aims to investigate the effect of pH on bacterial growth and diversity between upstream and downstream sediment sites. Methods used include bacteria culture, DNA extraction and 16S rRNA sequencing to identify bacteria species, DAPI staining to measure bacteria abundance, and GC-MS and XRF to analyze organic and inorganic compounds. The results found differences in culturable bacteria, abundance, organic compounds, and elements between the upstream low pH site and downstream higher pH site impacted by contaminated surface water runoff.
This document discusses geochemical controls during the biodegradation of petroleum hydrocarbons in soil. It provides background on past research showing microorganisms can use iron reduction to generate energy. More recent research has found biological iron redox transformations greatly influence aquifer and groundwater geochemistry. The document proposes a study to investigate intrinsic, iron-mediated degradation of volatile hydrocarbons like toluene in contaminated subsurface environments. It involves three stages: 1) laboratory experiments under anoxic conditions to simulate contaminated soil, 2) chemical extractions to assess iron pools involved in contaminant removal, and 3) stable carbon isotope analysis to differentiate carbonate pools and elucidate the effect of soil matrices on toluene removal and carbon
This document discusses using phosphate oxygen isotope ratios (δ18OP) to better understand phosphorus cycling in agricultural soils. It presents the goals of developing δ18OP as a tracer to identify the bioavailable P fraction in soils and track the long-term fate of externally applied P. It describes sample processing methods, measurement techniques, and initial findings showing transformation of fertilizer P into recalcitrant apatite P pools in agricultural soils. The document concludes that stable isotope labeling and tracking allows a deeper understanding of P sources, transfer, and transformations in natural environments.
Biological Nitrogen Fixation
Contents:
Introduction
Methods for measuring N2 fixation
1. Ntrogen balance method
2. Nitrogen difference method
3. Ureides method
4.〖𝟏𝟓〗_𝑵 isotope techniques
5. Acetylene reduction assay
6. Hydrogen evolution method
Introduction
N2 gas are found 78.084%on atmosphere of earth.
Nitrogen is an essential element for plant growth and development and a key issue of agriculture.
N2 are found in molecular N2 (𝑵 ≡ 𝑵) form in soil.
Dinitrogen is more stable, so we need of nitrogen fixation.
Most studies indicate that nitrogen fertilizers contribute to resolving the challenge the world is facing, feeding the human population.
The Green revolution was accompanied by an enormous increase in the application of nitrogen fertilizer.
Nitrogen fixation is a process by which nitrogen of the Earth's atmosphere is converted into ammonia (NH3), nitrogen salts or other molecules available to living organisms.
Biological Nitrogen Fixation(BNF) is known to be a sustain agriculture and increase soil fertility.
Research on microorganisms and plants able to fix nitrogen contributes largely to the production of bio fertilizers.
Thus it is important to ensure that BNF research and development will take into account the needs of farmers in the developing countries mainly.
Role of nitrogen in Plant
Sources of Nitrogen
Why measure 𝑵_𝟐 fixation?
Ecological consideration require an understanding of the relative contribution of 𝑵_𝟐 fixing components to the N-cycle.
Measurement of 𝑁_2 fixation enable an investigator to evaluate the ability of indigenous Rhizobium spp. to effectively nodulate newly introduced legumes.
Development of sustainable farming systems.
Understanding of the amount of 𝑵_𝟐fixed by legumes as influenced by soil management or cultural practices allows development of efficient agricultural and agroforesty production systems.
Performance of Fluidized Bed Biofilm Reactor for Nitrate RemovalIJRES Journal
Nitrate is present in the majority of water resources, and has reached serious level in many parts of the world, which is responsible for environmental problems. Hence it is necessary to remove nitrate. Biological denitrification provides the most economical means for nitrate removal. This paper represents the performance of Fluidized Bed Biofilm Reactor (FBBR) using bone china fine granules as biofilm carrier media for biological denitrification.
In this experimental work, the maximum average nitrogen removal efficiency of 93.71% at HRT of 30 minutes and optimum efficiency of 88.13% at HRT of 10 minutes is observed. For nitrogen loading rates varying from 0.48 to 28.80 kg N m-3 d-1, denitrification rates observed are 0.44 kg N m-3 d-1 to 17.26 kg N m-3 d-1. Optimum nitrogen loading rate and denitrification rate observed are 10.08 kg N m-3 d-1 and 8.88 kg N m-3 d-1 respectively. The results justify the usefulness of FBBR for denitrification.
Bioremediation of heavy metals using Fe(III),SULPHATE AND SULPHUR reducing ba...KAVYA K N
Bioremediation of heavy metals with the help of Fe(III),Sulfate AND Sulfur reducing bacteria bacteria,environmental clean up process using geobacter and desulfuromonas species.
Measuring Trace Concentrations of Phosphorus in Lake Auburn, ME using UV-Visi...Hannah Kiesler
This document presents a year-long thesis project analyzing methods to measure trace concentrations of phosphorus in Lake Auburn, Maine. The introduction provides background on the global phosphorus cycle and its importance in aquatic ecosystems. It describes how phosphorus naturally cycles through soil weathering and uptake by plants and animals, and how human activities have altered these cycles. Two analytical chemistry methods are tested: UV-visible spectrophotometry using the phosphomolybdate blue complex reaction, and chemiluminescence. The results suggest the chemiluminescence method provides better accuracy for measuring low phosphorus concentrations in freshwater samples like Lake Auburn.
Meulepas, 2009, Effect Of Environmental Conditions On Sulfate Reduction With ...roelmeulepas
1) The document describes experiments assessing the potential for using anaerobic oxidation of methane (AOM) coupled to sulfate reduction as an electron donor for biological sulfate reduction in industrial applications.
2) An enrichment of methane-oxidizing sulfate-reducing microorganisms from Eckernforde Bay sediment was used to study the effect of various environmental conditions on the rate of AOM and sulfate reduction.
3) The optimum conditions for AOM and sulfate reduction by the enrichment were a pH of 7.5, salinity of 30‰, and temperature of 20°C. AOM was completely inhibited at sulfide concentrations above 2.4 mM.
Bioremediation of toxic heavy metals (th ms) contaminated sites.2020Yahsé Challa
This document provides a review of bioremediation techniques for removing toxic heavy metals (THMs) such as arsenic, cadmium, chromium, mercury, and lead from contaminated sites. It discusses several microbial processes including oxidation-reduction, biomineralization, bioprecipitation, bioleaching, biosurfactant production, biovolatilization, biosorption, and bioaccumulation that can be harnessed for bioremediation. These processes take advantage of microbe-metal interactions like redox reactions, precipitation, and sorption to immobilize or remove THMs. The review also covers applications of these techniques in ex situ and in situ settings and potential improvements using genetic engineering and nanotechnology
1) The study investigated how seasonal temperature changes affected the release of dissolved organic carbon (DOC) and phenols from soils treated with different plant litter types over 22 months.
2) The researchers found that seasonal soil temperature positively correlated with DOC loss from litter-free soils, but this correlation diminished over time for soils treated with grass and ash litter.
3) Phenol concentrations in the senescing litters were small, resulting in minor losses to water. The variety of dissolved phenols in litter-amended soil leachates increased over time and was influenced by litter type.
This document compares methods for extracting and quantifying water-transportable phenols from different land uses. Solid phase extraction (SPE) was used to extract dissolved organic matter from water samples collected from grassland, woodland, and moorland sites. SPE followed by thermally assisted hydrolysis and methylation with tetramethylammonium hydroxide (THM) was shown to be an effective approach for isolating and quantifying lignin-derived phenols. All the different land uses exported similar amounts of lignin per unit weight of organic carbon in the drainage water. A significant proportion of lignin phenols may be lost from soils in particulate form, so total phenol loss is likely greater than previously estimated from dissolved phenols
This study examined the effects of iron and light stress on the pigment composition of an Antarctic Phaeocystis species. The study found that under low light, cells increased their content of chlorophyll a and light-harvesting pigments. Iron limitation decreased all light-harvesting pigments but cell volume also decreased, keeping cellular pigment concentrations similar. Under low light and iron limitation, chlorophyll a concentrations were suppressed. Ratios of light-harvesting pigments to chlorophyll a were highest under iron limitation. Iron stress also activated the photoprotective diatoxanthin cycle under high light and increased the diatoxanthin to diadinoxanthin ratio. Iron limitation induced synthesis of
This document provides an overview of bioremediation of metal contaminated soil. It discusses the sources of metal contamination in soil, the principles and types of bioremediation including in-situ and ex-situ techniques. It also describes the microorganisms used in bioremediation such as bacteria, fungi and algae, and the mechanisms involved including biosorption, bioimmobilization, bioleaching and biomineralization. Additionally, it covers phytoremediation techniques using plants and plant-microbe interactions in rhizoremediation. Designer microbe approaches for genetically engineered bioremediating organisms are also outlined.
his study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high-throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/L level after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing bacteria, the performance of these bioreactors had no significant difference between summer and winter.
Artifi cial wetlands are useful for wastewater treatment; however, relatively little is known of the effects of sewage on artifi cial wetland microbial community structure. Therefore, we assessed the effect of municipal sewage on microbial community diversity in surface water throughout an artifi cial wetland (Xiantao artifi cial wetland) treating municipal sewage. We analyzed the relationship between physicochemical parameters of surface water (i.e., Chemical Oxygen Demand (COD), Total Nitrogen (TN), Total Phosphorus (TP), and
NH4+-N) with microbial community structure (Illumina MiSeq sequencing followed by abundance indices). The results showed that the total microbial community in surface water was signifi cantly correlated with COD, TN, TP, and NH4
+-N (r = 0.764, 0.897, 0.883, 0.839, P < 0.05). In addition, the most abundant taxa were significantly correlated with COD (r = 0.803, P < 0.05). The relative abundance of rare operational taxonomic units in the more purifi ed water farther downstream was higher than in the polluted area, suggesting that rare groups were more sensitive to physicochemical parameters than abundant groups, and that the abundance of some bacteria could indirectly indicate the degree of aquatic pollution. Our results indicate that the responses of microorganisms in artificial wetlands to environmental conditions should be considered to ensure efficient treatment.
Phytoextraction, also called phytoaccumulation, phytoabsorption, or phytosequestration, refers to the use of plants to absorb, translocate, and store toxic contaminants from soil, sediments, and/or sludge in the root and shoot tissues .
Lead is an extremely difficult soil contaminant to remediate because it is a “soft” Lewis acid that forms strong bonds to both organic and inorganic ligands in soil. For the most part, Pb-contaminated soils are remediated through civil engineering techniques that require the excavation and landfilling of the contaminated soil. Soils that present a leaching hazard in the landfill are either placed in a specially constructed hazardous waste landfill, or treated with stabilizing agents, such as cement, prior to disposal in an industrial landfill.
This document describes research on nitrogen transformations in aquaponic systems. The objectives were to quantify the impact of physical and chemical variables on nitrogen transformations, evaluate transformations of different nitrogen forms under various conditions, examine ecologically important species, and investigate greenhouse gas emissions. Experiments were conducted with different hydraulic loading rates, pH levels, plant species, and sediment draining frequencies. The results showed that nitrate accumulation was affected by loading rate, pH, and plant uptake rate, while nitrogen loss occurred through denitrification. Microbial analyses provided insight into nitrifying bacteria populations and their role in the nitrogen cycle.
This document discusses bioremediation and the use of microorganisms to degrade organic pollutants and remove contamination. It describes how bacteria, fungi and other microbes break down waste organic matter through metabolic processes. The document also discusses how genetic engineering can be used to design microorganisms capable of degrading specific contaminants more efficiently. Examples are provided of various bacteria and fungi that have been genetically modified or studied for their ability to break down pollutants like benzene, toluene, chlorobenzoate and heavy metals.
Heavy metal contamination of soil is one of the most important environmental ...Ahmed Madni
This document discusses various heavy metal removal processes from soil and wastewater. It describes techniques for soil remediation including immobilization, soil washing, phytoremediation, and electroremediation. For wastewater treatment, it outlines chemical precipitation, ion exchange, adsorption, membrane filtration, coagulation/flocculation, and flotation. The key advantages and disadvantages of each technique are provided.
Abstract Heavy metal pollutants are mainly derived from growing number of
anthropogenic sources. As the environmental pollution with heavy metals increases,
some new technologies are being developed, one of these being phytoremediation.
Hyperaccumulator plant varieties can be achieved by using methods of genetic
engineering. An uptake of excessive amounts of heavy metals by plants from soil
solution leads to range of interactions at cellular level which produce toxic effects
on cell metabolism in terms of enzyme activity, protein structure, mineral nutrition,
water balance, respiration and ATP content, photosynthesis, growth and morphogenesis
and formation of reactive oxygen species (ROS).On the basis of accumulation
of heavy metals plants are divided into three main types; (i) the accumulator plants,
(ii) the indicator plants, and (iii) the excluder plants. Generally, the accumulation
of heavy metals in plant organ is in series root > leaves > stem > inflorescenc >
seed. Most of plants belong to excluder group and accumulate heavy metals in
their underground parts. When roots absorb heavy metals, they accumulate primarily
in rhizodermis and cortex. In intracellular parts, highest concentration of
heavy metals is found in cell wall. Tolerance of plants against heavy metals is due
to reduced uptake of heavy metals and increased plant internal sequestration. In the
increased plant internal sequestration mechanism, plant is manifested by interaction
between a genotype and its environment. There are biochemical machineries in
plants that work for tolerance and accumulation of heavy metals. Metal transporters
are involved in metal ion homeostasis and transportation. Some amino acids and
organic acids are ligands for heavy metals and these amino acids and organic acids
play an important role in tolerance and detoxification Phytochelatins (PCs) are produced
in plants under stress of heavy metals and play role in binding heavy metals
to complexes and salts and sequestering the compounds inside the cell so that heavy
metals can not disturb the cell metabolism. The genes for phytochelatin synthesis
have been isolated and characterized. Another low molecular weight (6–7 KDa)
cysteine-rich compounds known as metallothioneins (MTs) also play an important proteins, lipids, and nucleic acids, often leading to alterations in cell structure and
mutagenesis. There are many potential sources of ROS in plants, in addition to those
that come from reactions involved in normal metabolism, such as photosynthesis
and respiration. The balance between the steady-state levels of different ROS are
determined by the interplay between different ROS-producing and ROS-scavenging
mechanisms. A variety of proteins function as scavengers of superoxide and hydrogen
peroxide. These include, among others, superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), thioredoxin, and
the peroxiredoxin family of protein. These protein antioxidants are supplemented
with a host of non-protein scavengers, including, but not limited to, intracellular
ascorbate and glutathione. The intoxication with some heavy metals induces oxidative
stress because they are involved in several different types of ROS-generating
mechanisms.
This is a presentation I just did with about 40 of my 7th-9th grade cyber school students today. Some of them are still struggling to understand how to use many of the tools of our school system.
This document provides a high-level overview of major historical periods and events from the Renaissance through modern times, including religious movements like the Reformation, wars and conflicts such as the religious wars and World Wars, political developments like constitutionalism and absolutism, intellectual movements including the Scientific Revolution, Enlightenment, and Age of Isms, and social changes regarding women/family and art through the ages.
This document describes Demba Jammeh's research project exploring bacterial biodiversity in acid mine contaminated sediments in Adventdalen Landfill, Svalbard, Norway. The project aims to investigate the effect of pH on bacterial growth and diversity between upstream and downstream sediment sites. Methods used include bacteria culture, DNA extraction and 16S rRNA sequencing to identify bacteria species, DAPI staining to measure bacteria abundance, and GC-MS and XRF to analyze organic and inorganic compounds. The results found differences in culturable bacteria, abundance, organic compounds, and elements between the upstream low pH site and downstream higher pH site impacted by contaminated surface water runoff.
This document discusses geochemical controls during the biodegradation of petroleum hydrocarbons in soil. It provides background on past research showing microorganisms can use iron reduction to generate energy. More recent research has found biological iron redox transformations greatly influence aquifer and groundwater geochemistry. The document proposes a study to investigate intrinsic, iron-mediated degradation of volatile hydrocarbons like toluene in contaminated subsurface environments. It involves three stages: 1) laboratory experiments under anoxic conditions to simulate contaminated soil, 2) chemical extractions to assess iron pools involved in contaminant removal, and 3) stable carbon isotope analysis to differentiate carbonate pools and elucidate the effect of soil matrices on toluene removal and carbon
This document discusses using phosphate oxygen isotope ratios (δ18OP) to better understand phosphorus cycling in agricultural soils. It presents the goals of developing δ18OP as a tracer to identify the bioavailable P fraction in soils and track the long-term fate of externally applied P. It describes sample processing methods, measurement techniques, and initial findings showing transformation of fertilizer P into recalcitrant apatite P pools in agricultural soils. The document concludes that stable isotope labeling and tracking allows a deeper understanding of P sources, transfer, and transformations in natural environments.
Biological Nitrogen Fixation
Contents:
Introduction
Methods for measuring N2 fixation
1. Ntrogen balance method
2. Nitrogen difference method
3. Ureides method
4.〖𝟏𝟓〗_𝑵 isotope techniques
5. Acetylene reduction assay
6. Hydrogen evolution method
Introduction
N2 gas are found 78.084%on atmosphere of earth.
Nitrogen is an essential element for plant growth and development and a key issue of agriculture.
N2 are found in molecular N2 (𝑵 ≡ 𝑵) form in soil.
Dinitrogen is more stable, so we need of nitrogen fixation.
Most studies indicate that nitrogen fertilizers contribute to resolving the challenge the world is facing, feeding the human population.
The Green revolution was accompanied by an enormous increase in the application of nitrogen fertilizer.
Nitrogen fixation is a process by which nitrogen of the Earth's atmosphere is converted into ammonia (NH3), nitrogen salts or other molecules available to living organisms.
Biological Nitrogen Fixation(BNF) is known to be a sustain agriculture and increase soil fertility.
Research on microorganisms and plants able to fix nitrogen contributes largely to the production of bio fertilizers.
Thus it is important to ensure that BNF research and development will take into account the needs of farmers in the developing countries mainly.
Role of nitrogen in Plant
Sources of Nitrogen
Why measure 𝑵_𝟐 fixation?
Ecological consideration require an understanding of the relative contribution of 𝑵_𝟐 fixing components to the N-cycle.
Measurement of 𝑁_2 fixation enable an investigator to evaluate the ability of indigenous Rhizobium spp. to effectively nodulate newly introduced legumes.
Development of sustainable farming systems.
Understanding of the amount of 𝑵_𝟐fixed by legumes as influenced by soil management or cultural practices allows development of efficient agricultural and agroforesty production systems.
Performance of Fluidized Bed Biofilm Reactor for Nitrate RemovalIJRES Journal
Nitrate is present in the majority of water resources, and has reached serious level in many parts of the world, which is responsible for environmental problems. Hence it is necessary to remove nitrate. Biological denitrification provides the most economical means for nitrate removal. This paper represents the performance of Fluidized Bed Biofilm Reactor (FBBR) using bone china fine granules as biofilm carrier media for biological denitrification.
In this experimental work, the maximum average nitrogen removal efficiency of 93.71% at HRT of 30 minutes and optimum efficiency of 88.13% at HRT of 10 minutes is observed. For nitrogen loading rates varying from 0.48 to 28.80 kg N m-3 d-1, denitrification rates observed are 0.44 kg N m-3 d-1 to 17.26 kg N m-3 d-1. Optimum nitrogen loading rate and denitrification rate observed are 10.08 kg N m-3 d-1 and 8.88 kg N m-3 d-1 respectively. The results justify the usefulness of FBBR for denitrification.
Bioremediation of heavy metals using Fe(III),SULPHATE AND SULPHUR reducing ba...KAVYA K N
Bioremediation of heavy metals with the help of Fe(III),Sulfate AND Sulfur reducing bacteria bacteria,environmental clean up process using geobacter and desulfuromonas species.
Measuring Trace Concentrations of Phosphorus in Lake Auburn, ME using UV-Visi...Hannah Kiesler
This document presents a year-long thesis project analyzing methods to measure trace concentrations of phosphorus in Lake Auburn, Maine. The introduction provides background on the global phosphorus cycle and its importance in aquatic ecosystems. It describes how phosphorus naturally cycles through soil weathering and uptake by plants and animals, and how human activities have altered these cycles. Two analytical chemistry methods are tested: UV-visible spectrophotometry using the phosphomolybdate blue complex reaction, and chemiluminescence. The results suggest the chemiluminescence method provides better accuracy for measuring low phosphorus concentrations in freshwater samples like Lake Auburn.
Meulepas, 2009, Effect Of Environmental Conditions On Sulfate Reduction With ...roelmeulepas
1) The document describes experiments assessing the potential for using anaerobic oxidation of methane (AOM) coupled to sulfate reduction as an electron donor for biological sulfate reduction in industrial applications.
2) An enrichment of methane-oxidizing sulfate-reducing microorganisms from Eckernforde Bay sediment was used to study the effect of various environmental conditions on the rate of AOM and sulfate reduction.
3) The optimum conditions for AOM and sulfate reduction by the enrichment were a pH of 7.5, salinity of 30‰, and temperature of 20°C. AOM was completely inhibited at sulfide concentrations above 2.4 mM.
Bioremediation of toxic heavy metals (th ms) contaminated sites.2020Yahsé Challa
This document provides a review of bioremediation techniques for removing toxic heavy metals (THMs) such as arsenic, cadmium, chromium, mercury, and lead from contaminated sites. It discusses several microbial processes including oxidation-reduction, biomineralization, bioprecipitation, bioleaching, biosurfactant production, biovolatilization, biosorption, and bioaccumulation that can be harnessed for bioremediation. These processes take advantage of microbe-metal interactions like redox reactions, precipitation, and sorption to immobilize or remove THMs. The review also covers applications of these techniques in ex situ and in situ settings and potential improvements using genetic engineering and nanotechnology
1) The study investigated how seasonal temperature changes affected the release of dissolved organic carbon (DOC) and phenols from soils treated with different plant litter types over 22 months.
2) The researchers found that seasonal soil temperature positively correlated with DOC loss from litter-free soils, but this correlation diminished over time for soils treated with grass and ash litter.
3) Phenol concentrations in the senescing litters were small, resulting in minor losses to water. The variety of dissolved phenols in litter-amended soil leachates increased over time and was influenced by litter type.
This document compares methods for extracting and quantifying water-transportable phenols from different land uses. Solid phase extraction (SPE) was used to extract dissolved organic matter from water samples collected from grassland, woodland, and moorland sites. SPE followed by thermally assisted hydrolysis and methylation with tetramethylammonium hydroxide (THM) was shown to be an effective approach for isolating and quantifying lignin-derived phenols. All the different land uses exported similar amounts of lignin per unit weight of organic carbon in the drainage water. A significant proportion of lignin phenols may be lost from soils in particulate form, so total phenol loss is likely greater than previously estimated from dissolved phenols
This study examined the effects of iron and light stress on the pigment composition of an Antarctic Phaeocystis species. The study found that under low light, cells increased their content of chlorophyll a and light-harvesting pigments. Iron limitation decreased all light-harvesting pigments but cell volume also decreased, keeping cellular pigment concentrations similar. Under low light and iron limitation, chlorophyll a concentrations were suppressed. Ratios of light-harvesting pigments to chlorophyll a were highest under iron limitation. Iron stress also activated the photoprotective diatoxanthin cycle under high light and increased the diatoxanthin to diadinoxanthin ratio. Iron limitation induced synthesis of
This document provides an overview of bioremediation of metal contaminated soil. It discusses the sources of metal contamination in soil, the principles and types of bioremediation including in-situ and ex-situ techniques. It also describes the microorganisms used in bioremediation such as bacteria, fungi and algae, and the mechanisms involved including biosorption, bioimmobilization, bioleaching and biomineralization. Additionally, it covers phytoremediation techniques using plants and plant-microbe interactions in rhizoremediation. Designer microbe approaches for genetically engineered bioremediating organisms are also outlined.
his study investigated the microbial community in a full scale anaerobic baffled reactor and sequencing batch reactor system for oil-produced water treatment in summer and winter. The community structures of fungi and bacteria were analyzed through polymerase chain reaction–denaturing gradient gel electrophoresis and Illumina high-throughput sequencing, respectively. Chemical oxygen demand effluent concentration achieved lower than 50 mg/L level after the system in both summer and winter, however, chemical oxygen demand removal rates after anaerobic baffled reactor treatment system were significant higher in summer than that in winter, which conformed to the microbial community diversity. Saccharomycotina, Fusarium, and Aspergillus were detected in both anaerobic baffled reactor and sequencing batch reactor during summer and winter. The fungal communities in anaerobic baffled reactor and sequencing batch reactor were shaped by seasons and treatment units, while there was no correlation between abundance of fungi and chemical oxygen demand removal rates. Compared to summer, the total amount of the dominant hydrocarbon degrading bacteria decreased by 10.2% in anaerobic baffled reactor, resulting in only around 23% of chemical oxygen demand was removed in winter. Although microbial community significantly varied in the three parallel sulfide reducing bacteria, the performance of these bioreactors had no significant difference between summer and winter.
Artifi cial wetlands are useful for wastewater treatment; however, relatively little is known of the effects of sewage on artifi cial wetland microbial community structure. Therefore, we assessed the effect of municipal sewage on microbial community diversity in surface water throughout an artifi cial wetland (Xiantao artifi cial wetland) treating municipal sewage. We analyzed the relationship between physicochemical parameters of surface water (i.e., Chemical Oxygen Demand (COD), Total Nitrogen (TN), Total Phosphorus (TP), and
NH4+-N) with microbial community structure (Illumina MiSeq sequencing followed by abundance indices). The results showed that the total microbial community in surface water was signifi cantly correlated with COD, TN, TP, and NH4
+-N (r = 0.764, 0.897, 0.883, 0.839, P < 0.05). In addition, the most abundant taxa were significantly correlated with COD (r = 0.803, P < 0.05). The relative abundance of rare operational taxonomic units in the more purifi ed water farther downstream was higher than in the polluted area, suggesting that rare groups were more sensitive to physicochemical parameters than abundant groups, and that the abundance of some bacteria could indirectly indicate the degree of aquatic pollution. Our results indicate that the responses of microorganisms in artificial wetlands to environmental conditions should be considered to ensure efficient treatment.
Phytoextraction, also called phytoaccumulation, phytoabsorption, or phytosequestration, refers to the use of plants to absorb, translocate, and store toxic contaminants from soil, sediments, and/or sludge in the root and shoot tissues .
Lead is an extremely difficult soil contaminant to remediate because it is a “soft” Lewis acid that forms strong bonds to both organic and inorganic ligands in soil. For the most part, Pb-contaminated soils are remediated through civil engineering techniques that require the excavation and landfilling of the contaminated soil. Soils that present a leaching hazard in the landfill are either placed in a specially constructed hazardous waste landfill, or treated with stabilizing agents, such as cement, prior to disposal in an industrial landfill.
This document describes research on nitrogen transformations in aquaponic systems. The objectives were to quantify the impact of physical and chemical variables on nitrogen transformations, evaluate transformations of different nitrogen forms under various conditions, examine ecologically important species, and investigate greenhouse gas emissions. Experiments were conducted with different hydraulic loading rates, pH levels, plant species, and sediment draining frequencies. The results showed that nitrate accumulation was affected by loading rate, pH, and plant uptake rate, while nitrogen loss occurred through denitrification. Microbial analyses provided insight into nitrifying bacteria populations and their role in the nitrogen cycle.
This document discusses bioremediation and the use of microorganisms to degrade organic pollutants and remove contamination. It describes how bacteria, fungi and other microbes break down waste organic matter through metabolic processes. The document also discusses how genetic engineering can be used to design microorganisms capable of degrading specific contaminants more efficiently. Examples are provided of various bacteria and fungi that have been genetically modified or studied for their ability to break down pollutants like benzene, toluene, chlorobenzoate and heavy metals.
Heavy metal contamination of soil is one of the most important environmental ...Ahmed Madni
This document discusses various heavy metal removal processes from soil and wastewater. It describes techniques for soil remediation including immobilization, soil washing, phytoremediation, and electroremediation. For wastewater treatment, it outlines chemical precipitation, ion exchange, adsorption, membrane filtration, coagulation/flocculation, and flotation. The key advantages and disadvantages of each technique are provided.
Abstract Heavy metal pollutants are mainly derived from growing number of
anthropogenic sources. As the environmental pollution with heavy metals increases,
some new technologies are being developed, one of these being phytoremediation.
Hyperaccumulator plant varieties can be achieved by using methods of genetic
engineering. An uptake of excessive amounts of heavy metals by plants from soil
solution leads to range of interactions at cellular level which produce toxic effects
on cell metabolism in terms of enzyme activity, protein structure, mineral nutrition,
water balance, respiration and ATP content, photosynthesis, growth and morphogenesis
and formation of reactive oxygen species (ROS).On the basis of accumulation
of heavy metals plants are divided into three main types; (i) the accumulator plants,
(ii) the indicator plants, and (iii) the excluder plants. Generally, the accumulation
of heavy metals in plant organ is in series root > leaves > stem > inflorescenc >
seed. Most of plants belong to excluder group and accumulate heavy metals in
their underground parts. When roots absorb heavy metals, they accumulate primarily
in rhizodermis and cortex. In intracellular parts, highest concentration of
heavy metals is found in cell wall. Tolerance of plants against heavy metals is due
to reduced uptake of heavy metals and increased plant internal sequestration. In the
increased plant internal sequestration mechanism, plant is manifested by interaction
between a genotype and its environment. There are biochemical machineries in
plants that work for tolerance and accumulation of heavy metals. Metal transporters
are involved in metal ion homeostasis and transportation. Some amino acids and
organic acids are ligands for heavy metals and these amino acids and organic acids
play an important role in tolerance and detoxification Phytochelatins (PCs) are produced
in plants under stress of heavy metals and play role in binding heavy metals
to complexes and salts and sequestering the compounds inside the cell so that heavy
metals can not disturb the cell metabolism. The genes for phytochelatin synthesis
have been isolated and characterized. Another low molecular weight (6–7 KDa)
cysteine-rich compounds known as metallothioneins (MTs) also play an important proteins, lipids, and nucleic acids, often leading to alterations in cell structure and
mutagenesis. There are many potential sources of ROS in plants, in addition to those
that come from reactions involved in normal metabolism, such as photosynthesis
and respiration. The balance between the steady-state levels of different ROS are
determined by the interplay between different ROS-producing and ROS-scavenging
mechanisms. A variety of proteins function as scavengers of superoxide and hydrogen
peroxide. These include, among others, superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), thioredoxin, and
the peroxiredoxin family of protein. These protein antioxidants are supplemented
with a host of non-protein scavengers, including, but not limited to, intracellular
ascorbate and glutathione. The intoxication with some heavy metals induces oxidative
stress because they are involved in several different types of ROS-generating
mechanisms.
This is a presentation I just did with about 40 of my 7th-9th grade cyber school students today. Some of them are still struggling to understand how to use many of the tools of our school system.
This document provides a high-level overview of major historical periods and events from the Renaissance through modern times, including religious movements like the Reformation, wars and conflicts such as the religious wars and World Wars, political developments like constitutionalism and absolutism, intellectual movements including the Scientific Revolution, Enlightenment, and Age of Isms, and social changes regarding women/family and art through the ages.
The document describes the identification, cloning, sequencing, and characterization of the a-L-arabinofuranosidase B (abfB) gene from the phytopathogenic fungus Fusarium oxysporum f. sp. dianthi (Fod). The gene was identified using random amplified polymorphic DNA and encodes a protein of 499 amino acids. The recombinant protein expressed in E. coli had arabinofuranosidase activity and optimal activity at pH 4.0 and 50°C. Reverse transcription PCR showed that the abfB gene is actively transcribed in carnation plants infected with Fod, suggesting it plays a role in the fungus's pathogenicity.
Why Doesn't My Website Show Up In The Search Engines?shuey03
The document discusses why websites may not show up in search engines and provides tips for optimization. It explains that search engines see websites differently than humans and can be impacted by technical issues like robots.txt files and duplicate content. The tips discussed include structuring content in a clear hierarchy with internal links, creating valuable blog posts on relevant topics and optimizing them for keywords, and building links and social shares through strategies like guest blogging, social sharing, and creating helpful resources. The goal is to make websites easily accessible to both humans and search engines.
This document summarizes research on the induction of laccase gene expression in the white-rot fungus Trametes sp. I-62 by molasses wastewaters and melanoidins. The key findings are:
1) Molasses wastewaters and isolated melanoidins induced increased laccase activity and selective expression of the lcc1 and lcc2 laccase genes in fungal cultures.
2) The highest laccase activity and earliest induction of laccase genes occurred when cultures were exposed to complete molasses wastewater compared to isolated melanoidins.
3) Decolorization of molasses wastewater by the fungus closely correlated with increased laccase activity
1. Several Italian city-states like Venice, Genoa, Milan, and Florence grew extremely wealthy through overseas trade in the 12th-15th centuries, fueling economic growth and patronage of the arts.
2. Many northern Italian cities established communes ruled by merchant oligarchies, though the common people sought more political power through popular uprisings.
3. By the 15th century, Italy was dominated by several powerful states including Venice, Milan, Florence, the Papal States, and Naples, who worked to maintain a balance of power to prevent any one from gaining complete control. However, the failure to unify made Italy vulnerable to foreign invasion and conquest.
This is the slide deck of how I got about building links in real time. It's a creative method of link building that takes some effort, but the results and phenomenal.
This document discusses the history and development of using soil microbial biomass as an indicator of environmental impacts on soil ecosystems. In the 1980s, ecosystem research projects aimed to quantify the effects of pollution. Researchers realized the soil microbe compartment was important for nutrient cycling. Direct observation methods were inadequate, but the chloroform fumigation incubation technique allowed quantifying total microbial biomass. The authors then developed using maximum CO2 output of soils in relation to microbial biomass to assess environmental stress on soil microbes. This built on prior work establishing the concept of microbial maintenance energy, the energy microbes require to maintain themselves even without growth.
Eight new primer sets were designed to detect genes involved in bacterial aerobic BTEX degradation through PCR. The primer sets target genes encoding mono-oxygenases and dioxygenases for the initial attack of BTEX, as well as catechol 2,3-dioxygenase genes responsible for ring cleavage. The primers allowed detection of the corresponding genes in soil with detection limits of 103-104 or 105-106 gene copies per gram of soil. The primer sets were used to assess the distribution of catabolic genes in BTEX-degrading bacterial isolates and DNA from soils at a BTEX-contaminated site. tmoA-, xylM- and xylE1-like genes were most frequently detected in
Microbial community composition of different soil layers in an aged oil spill...Erhovwon Aggreh
The document summarizes a study on the microbial community composition of different soil layers from an aged oil spill site in Nigeria. Total petroleum hydrocarbons and polycyclic aromatic hydrocarbons were higher in the top soil compared to the subsoil. A total of 24 bacterial species from 11 genera and 10 fungal species from 7 genera were isolated from both soil layers and confirmed to degrade oil. The top soil contained Proteus, Salmonella, Citrobacter, Enterobacter, Klebsiella, Bacillus and Corynebacterium, while the subsoil contained Escherichia, Flavobacterium, Corynebacterium, Pseudomonas, and Bacillus. Gammaproteobacteria
Jagerma, 2009, Microbial Diversity And Community Structure Of A Highly Active...roelmeulepas
This study characterized the microbial community in a bioreactor enrichment capable of high-rate anaerobic methane oxidation (AOM) coupled to sulfate reduction. Analysis showed that methanotrophy was performed predominantly by archaea belonging to the ANME-2a subgroup. Sulfate reduction was likely carried out by deltaproteobacterial sulfate-reducing bacteria related to Desulfotignum. Incubation with 13C-labeled methane revealed substantial incorporation of 13C into both bacterial and archaeal lipids, indicating active methane consumption by both groups. While some ANME-2/sulfate-reducer consortia were present, many ANME-2a cells occurred as single cells not closely associated with bacterial partners. This
1. Vermicomposting is a process that uses earthworms to convert organic wastes into a nutrient-rich compost called vermicast or vermicompost. Earthworms feed on organic matter and excrete nutrient-dense castings.
2. The process involves collecting organic wastes and mixing them with materials like soil, leaves, and cow dung in a bin. Earthworms are then added and they feed and aerate the mixture as it decomposes.
3. After several weeks, the mixture is converted into vermicompost which is high in nutrients like nitrogen, phosphorus and potassium and improves soil fertility and plant growth when used.
Heavy metals and acid rain were applied over 4 years to a model forest ecosystem to study their effects on soil microbial communities. Microbial biomass, basal respiration, and hydrolase enzyme activities were reduced by heavy metal contamination, especially in combination with acid rain. Analysis of phospholipid fatty acids and genetic fingerprints showed that heavy metals altered the structure of bacterial communities over time. While soil pH and available heavy metal levels were similar between treatments with just heavy metals and those with both heavy metals and acid rain, the combined treatment had more drastic negative effects on the microbial community. This suggests that acid rain exacerbates the toxicity of heavy metals to soil microorganisms.
1) Soil microbes play an important role in global carbon and nitrogen cycles by driving processes like organic matter decomposition, nitrogen fixation, and methane/nitrous oxide emissions.
2) Changing environmental conditions due to climate change, like increased temperature and altered precipitation, can impact soil microbial communities and gene expression.
3) Horizontal gene transfer between soil microbes may be a natural adaptation strategy to environmental changes, allowing microbes to acquire new genes that help them survive. Studying this process of natural transformation could provide insights into molecular-level climate change adaptation.
Lagoas de alta taxa versando sobre avanços e princiais beneficios da tecnologiaLucasVassalledeCastr
This document summarizes a research project on using high rate algal ponds (HRAP) for post-treating sewage effluent from upflow anaerobic sludge blanket (UASB) reactors. UASB reactors are commonly used for sewage treatment in Brazil and other Latin American countries due to their lower costs compared to other technologies. However, UASB effluent does not always meet discharge standards for biochemical oxygen demand, nutrients, and pathogens. The research aims to evaluate whether HRAP can provide tertiary treatment of UASB effluent through anaerobic co-digestion of algal biomass, and further removal of nutrients and micropollutants, while improving sustainability.
Numerical simulation of bioremediation of poly aromatic hydrocarbon pollutedIAEME Publication
This document presents a numerical simulation of bioremediation of polyaromatic hydrocarbon (PAH) polluted soil using different mushroom species and MATLAB. Soil samples were divided into cells and treated with saprophytic, symbiotic, or parasitic mushroom substrate over 10 weeks. PAH concentration was measured every 2 weeks. A kinetic model was developed and rate constants were calculated using the integral method in MATLAB. Results showed the parasitic mushroom degraded PAHs fastest with a rate constant of 0.3751 day-1, followed by symbiotic and saprophytic mushrooms. This indicates mushroom-assisted bioremediation can effectively degrade PAH pollution in soil over time.
LABORATORY STUDIES ON THE BIOREMEDIATION OF SOIL CONTAMINATED BY DIESEL IAEME Publication
The most widely used energy and fuel resources are hydrocarbons such as crude oil and petroleum distillates. The accidental discharge of these petroleum products contribute in making hydrocarbons the most common environmental pollutants. Bioremediation helps to destroy or render harmless various contaminants using natural biological activity. The present study utilizes the potential of bioremediation to remediate soil contaminated with diesel. Eight bioreactors were used for the study, out of which four bioreactors were maintained at optimum environmental conditions and the remaining four were kept without any maintenance to serve as control bioreactors. Contaminated soil was prepared by mixing fresh soil and diesel so as to attain 10% TPH concentrations by weight of soil. Each bioreactor was filled with 3 kg of contaminated soil.
Acceleration of Lead Phytostabilization by Maize (Zea mays) in Association wi...Agriculture Journal IJOEAR
Abstract— Soil where shooting practices are conducted is extremely contaminated with heavy metals, exclusively by Pb, due to the bullets and bullet fragments. These extreme concentrations of Pb, generate an unfavourable surroundings for agriculture and human health through phytoaccumulation. This study was conducted to assess the possibility of particular bio-amendment, phytostabilization on the reduction of bioavailable Pb in such contaminated soil. Biomass (BM) produced from Gliricidiasepium was used to see its ability to be used in soil remediation together with Maize (Zea mays) as phytostabilizer. A pot experiment was conducted with Maizeby adding BM at three different percentages, 1, 2.5 and 5% (w/w). Soil without amendments served as the control and arranged in a complete randomized design. By maize, translocation rate of heavy metals into crop were determined. After sixth week, grown maize were harvested and analyzed followed by digestion with con.HNO3. The most significant immobilization (p<0.05)>TF in maize. For the treatment 5% BM, PF and TF for Pb are 1.22 and 0.15 respectively. Thereby maize can be considered as a potential phytostabilizer. At the same time efficiency of phytostabilizing nature of maizecan increase together with the application of soil amendment – BM.
This document is a thesis project examining innovative biological phosphate and anaerobic digestion technology for waste treatment, energy generation, and phosphorus recovery. It includes an abstract, introduction covering topics like sewage, current bioremediation methods, anaerobic digestion processes, low temperature anaerobic digestion, bioreactor configurations, and the global phosphorus crisis. It also outlines the materials and methods, expected results sections, and planned discussion. The introduction provides background on anaerobic digestion and examines its application to low-temperature wastewater treatment.
Bioremediating Effect of Glomus Hoi and Pseudomonas Aeruginosa on the Organic...IJEAB
This study analyzed the degrading effect of Glomus hoi and Pseudomonas aeruginosa on the organic content and heavy metals of oil refinery effluent polluted soil using Amaranthus cruentus as the test plant. This study was carried out to determine if agricultural activities can be improved using any or both of the micoorganisms. Eight different treatment layouts were used with three replicates for each level of pollution in the treatment layout. Ninety six (96) pots, each containing three kilograms of soil from both sterilized and unsterilized soil were used for the study. Fifty (50) grams of soil inoculum from propagated Arbuscular mycorrhiza was inoculated to a set of twenty four (24) experimental pots containing both sterilized and unsterilized soil before A. cruentus seedlings were transplanted to them. Another set of twenty four (24) pots containing both sterilized and unsterilized soil were injected with thirty (30) mL of P. aeruginosa inoculum solution before transplanting A. cruentus seedlings to them. The third set of twenty four (24) pots received dual inoculation of both fifty (50) grams of soil inoculum containing G. hoi and thirty (30) mL of P. aeruginosa inoculum solution before A. cruentus were transplanted to them. The residual twenty four (24) pots served as the control. Thereafter, pot preparation was arranged in the screenhouse in a randomized block design. The A. cruentus seedlings were raised in nursery for a period of two weeks before they were transplanted to the pots, seedlings were left for 3 days to overcome transplanting shock before contaminating the soil with refinery effluent at various concentrations of 0%, 2%, 4% and 6% v/w. The seedlings were allowed to grow for eight weeks before the termination of the experiment. The pre planting analysis of soil showed that heavy metals analyses (zinc and iron) of sterilized soil had a lower concentration to the unsterilized. The soil pH ranged from 6.3 to 6.8. It also revealed that organic matter and organic carbon content ranged from 0.8% to 1.3% and 0.4% to 1.7%. However, after the experiment, it was discovered in this study that treatments without any microorganism inoculation in sterilized and unsterilized soil had a higher level of % organic carbon and % organic matter content compared to the other treatments that were inoculated with one or two micro-organisms across all the levels of effluent concentration. Heavy metals of soil in all the soil samples were found to increase as the petrochemical effluent increased in concentration. The results obtained were analyzed using Duncan Multiple Range Test (DMRT) and other descriptive statistics. This study opined that the combined use of G. hoi and P. aeruginosa was more effective in improving the organic contentand the reduce heavy metals of oil refinery effluent polluted soil than when either is used singly.
COD reduction of aromatic polluted waste water by Advanced Oxidation Process ...Wade Bitaraf
In most petrochemical complexes and oil refineries the wastewater contains the aromatic compounds among which Benzene, Toluene, Ethyl Benzene and Xylene (BTEX) have harmful effects on environment and human health. The present work mainly deals with the UV-based advanced oxidation processes (AOPs), UV/H2O2 were tested in batch reactor systems to evaluate the removal efficiencies and optimal conditions for the photodegradation of BTEX in order to wastewater treatment. The efficiency of this method was analyzed by evaluating the Chemical Oxygen Demand (COD) as a pollution criterion through the COD reactor. The influence of the basic operational parameters such as initial concentration of H2O2, pH, Temperature, irradiation time and UV amount on the photo degradation of BTEX were also studied. The oxidation rate of BTEX and respectively the reduction rate of COD were low when the oxidation was carried out in the absence of H2O2 or UV light. The addition of proper amount of hydrogen peroxide improved the degradation, while the excess hydrogen peroxide could quench the formation of hydroxyl radicals (•OH). The optimal conditions of suspended slurry with 1.11(g/l) initial concentration of H2O2 and pH value of 3.1 were obtained under three UV lights illumination (6 W). Under the optimal conditions, COD reduction during the initial period of 180 min in UV/H2O2 systems reached about 90%.
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment as a result of incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic. While various physicochemical methods have been used to remove PAHs, they have limitations. Microorganisms have potential for bioremediation through degradation of PAHs, but their efficiency needs improvement. Research is exploring ways to enhance PAH degradation through metabolic engineering of microorganisms and optimizing factors like bioavailability and chemotaxis.
This document summarizes a research project on the effect of temperature on the bioavailability and microbial degradation of polycyclic aromatic hydrocarbons (PAHs) in soil. PAHs are toxic compounds found in the environment from both natural and industrial sources. The student aims to examine how temperature affects the bioavailability and biodegradation of two PAHs, fluoranthene and benzo[a]pyrene, in soil by conducting a microcosm experiment over different temperatures and analyzing samples after various time periods using gas chromatography. The results will provide information on how temperature influences PAH removal from soil to help design effective bioremediation strategies.
This document discusses various engineering strategies for bioremediation. It begins by outlining the importance of site characterization, pollutant characterization, and geohydrochemical characterization. It then discusses approaches like biotreatability tests, bioaugmentation, biopiling, biosparging, and different ex-situ techniques like land farming and composting. The key factors that affect bioremediation like nutrient requirements, oxygen supply, and mass transfer are also summarized.
This document discusses using soil washing with nonionic surfactants to remove phenanthrene (PHE) contamination from soil, and then using activated carbon to selectively adsorb the surfactants from the washed solution for recovery and reuse. Four nonionic surfactants - Tween 40, Tween 80, Brij 30 and Brij 35 - were tested for their ability to solubilize PHE and for their adsorption to soil. Brij 30 showed the highest solubilization of PHE and lowest adsorption to soil, making it most effective for soil washing. Activated carbon was then able to selectively adsorb 85-89% of the surfactants from the washed solutions, allowing recovery. However
Application Of Common Duckweed (Lemna Minor) In Phytoremediation Of Chemicals...Kathryn Patel
This document reviews the application of the common duckweed (Lemna minor) in phytoremediation of pollutants in the aquatic environment. It discusses how phytoremediation uses plants to remove pollutants from air, soil, and water. Aquatic macrophytes like duckweed are effective at remediating wastewater and various pollutants from water bodies. The document then focuses on the biology and uses of L. minor, describing how it has been used to remove organic pollutants, heavy metals, agricultural chemicals, pharmaceuticals, radioactive waste, and more from polluted water. It concludes by identifying gaps in research that need further study to advance ecological solutions for remediating polluted
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
The study aimed to detect and analyze novel diterpenoid dioxygenase genes (ditA1) involved in the degradation of resin acids, which are naturally produced by trees and released during wood pulping processes. Using newly designed PCR primers, ditA1 homolog genes were amplified from various Pseudomonas, Burkholderia, and Cupriavidus strains. All isolates containing a ditA1 homolog could grow on dehydroabietic acid and expressed ditA1 constitutively or in response to dehydroabietic acid, demonstrating their role in degradation. Evolutionary analyses indicate ditA1 and gyrB genes have coevolved from ancestral variants in Pseudomonas, Burkholderia, and Cupriavid
1) Tannic acid is able to chemically reduce the oxidized form of ABTS, the compound commonly used to detect laccase activity spectrophotometrically.
2) This chemical reduction decreases the detectable laccase activity values observed when laccase enzyme is present.
3) The reduction interferes with the laccase detection assay and can lead to underestimation of laccase activity in samples containing tannic acid or similar polyphenolic compounds.
This document summarizes a study that used PCR-SSCP analysis to assess the diversity of catechol 2,3-dioxygenase (C23O) genes, which encode key enzymes in aromatic hydrocarbon degradation pathways, in soils contaminated with BTEX (benzene, toluene, ethylbenzene and xylenes). PCR-SSCP generated site-specific fingerprints of C23O gene diversity that correlated with temporal changes and contamination levels. Predominant C23O gene variants were recovered and closely matched sequences from clone libraries. Isolates harboring genes identical to predominant variants mineralized benzene but not toluene, while an isolate with a single mutation in its C23O gene mineralized
This document describes a study that developed a new method called amplified functional DNA restriction analysis (AFDRA) to analyze the diversity of catechol 2,3-dioxygenase (C23O) genes in soil bacteria. C23O genes code for enzymes important for degrading aromatic pollutants. The researchers used AFDRA to analyze C23O genes from reference strains and soil isolates. They found that AFDRA generated distinct restriction patterns that clustered the isolates into four groups, consistent with sequence analysis. AFDRA also allowed them to determine the predominant C23O gene variants present in environmental DNA extracts from soil samples. The study demonstrates that AFDRA provides a rapid way to assess functional gene diversity in cultures and
This document describes research identifying a new laccase gene (lcc1A) in the fungus Trametes sp. I-62 using PCR-RFLP analysis. It confirms the intron/exon structures of three previously identified laccase genes (lcc1, lcc2, lcc3) by sequencing their cDNAs. Lcc1A showed 99.6% identity to lcc1 at the protein level, suggesting they are allelic variants. The identification of this laccase gene family contributes to understanding the biochemical diversity of laccases in fungi.
This document discusses the effect of tannic acid on laccase production in the white-rot fungus Coriolopsis gallica. The study found that adding tannic acid to the growth media increased extracellular laccase levels and laccase gene transcription in C. gallica. Specifically, concentrations of 50 and 100 μM tannic acid significantly increased laccase activity compared to control cultures without tannic acid. The results demonstrate that tannic acid induces laccase activity in C. gallica by enhancing expression of the laccase gene at the transcriptional level.
TrustArc Webinar - 2024 Global Privacy SurveyTrustArc
How does your privacy program stack up against your peers? What challenges are privacy teams tackling and prioritizing in 2024?
In the fifth annual Global Privacy Benchmarks Survey, we asked over 1,800 global privacy professionals and business executives to share their perspectives on the current state of privacy inside and outside of their organizations. This year’s report focused on emerging areas of importance for privacy and compliance professionals, including considerations and implications of Artificial Intelligence (AI) technologies, building brand trust, and different approaches for achieving higher privacy competence scores.
See how organizational priorities and strategic approaches to data security and privacy are evolving around the globe.
This webinar will review:
- The top 10 privacy insights from the fifth annual Global Privacy Benchmarks Survey
- The top challenges for privacy leaders, practitioners, and organizations in 2024
- Key themes to consider in developing and maintaining your privacy program
Why You Should Replace Windows 11 with Nitrux Linux 3.5.0 for enhanced perfor...SOFTTECHHUB
The choice of an operating system plays a pivotal role in shaping our computing experience. For decades, Microsoft's Windows has dominated the market, offering a familiar and widely adopted platform for personal and professional use. However, as technological advancements continue to push the boundaries of innovation, alternative operating systems have emerged, challenging the status quo and offering users a fresh perspective on computing.
One such alternative that has garnered significant attention and acclaim is Nitrux Linux 3.5.0, a sleek, powerful, and user-friendly Linux distribution that promises to redefine the way we interact with our devices. With its focus on performance, security, and customization, Nitrux Linux presents a compelling case for those seeking to break free from the constraints of proprietary software and embrace the freedom and flexibility of open-source computing.
Building RAG with self-deployed Milvus vector database and Snowpark Container...Zilliz
This talk will give hands-on advice on building RAG applications with an open-source Milvus database deployed as a docker container. We will also introduce the integration of Milvus with Snowpark Container Services.
UiPath Test Automation using UiPath Test Suite series, part 5DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 5. In this session, we will cover CI/CD with devops.
Topics covered:
CI/CD with in UiPath
End-to-end overview of CI/CD pipeline with Azure devops
Speaker:
Lyndsey Byblow, Test Suite Sales Engineer @ UiPath, Inc.
Cosa hanno in comune un mattoncino Lego e la backdoor XZ?Speck&Tech
ABSTRACT: A prima vista, un mattoncino Lego e la backdoor XZ potrebbero avere in comune il fatto di essere entrambi blocchi di costruzione, o dipendenze di progetti creativi e software. La realtà è che un mattoncino Lego e il caso della backdoor XZ hanno molto di più di tutto ciò in comune.
Partecipate alla presentazione per immergervi in una storia di interoperabilità, standard e formati aperti, per poi discutere del ruolo importante che i contributori hanno in una comunità open source sostenibile.
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Kabelitz Amb 2009 Online
1. Appl Microbiol Biotechnol
DOI 10.1007/s00253-009-1868-0
ENVIRONMENTAL BIOTECHNOLOGY
Enhancement of the microbial community biomass
and diversity during air sparging bioremediation of a soil
highly contaminated with kerosene and BTEX
Nadja Kabelitz & Jirina Machackova & Gwenaël Imfeld &
Maria Brennerova & Dietmar H. Pieper &
Hermann J. Heipieper & Howard Junca
Received: 6 October 2008 / Revised: 9 January 2009 / Accepted: 10 January 2009
# Springer-Verlag 2009
Abstract In order to obtain insights in complexity shifts quantities and composition of the microbial communities
taking place in natural microbial communities under strong developed at different stages of the bioventing treatment
selective pressure, soils from a former air force base in the progress was performed. Depending on the length of the air
Czech Republic, highly contaminated with jet fuel and at sparging treatment that led to a significant reduction in the
different stages of a bioremediation air sparging treatment, contamination level, we observed a clear shift in the soil
were analyzed. By tracking phospholipid fatty acids and microbial community being dominated by Pseudomonads
16S rRNA genes, a detailed monitoring of the changes in under the harsh conditions of high aromatic contamination
to a status of low aromatic concentrations, increased
biomass content, and a complex composition with diverse
Electronic supplementary material The online version of this article
bacterial taxonomical branches.
(doi:10.1007/s00253-009-1868-0) contains supplementary material,
which is available to authorized users.
Keywords BTEX . Air sparging . Bioremediation .
N. Kabelitz : H. J. Heipieper (*)
Department of Bioremediation, Biodiversity . Microbiota
Helmholtz Centre for Environmental Research (UFZ),
Permoserstr. 15,
04318 Leipzig, Germany Introduction
e-mail: hermann.heipieper@ufz.de
J. Machackova The spillage of organic compounds represents one of the
Earth Tech CZ s.r.o., biggest problems of contamination in soils and ground-
Trojská 92, water, especially in eastern European countries. Military
171 00 Prague 7, Czech Republic
areas particularly represent a major problem due to their
G. Imfeld high pollutant concentration. Therefore, massive attempts
Department of Isotope Biogeochemistry, are being carried out to remediate such sites, commonly
Helmholtz Centre for Environmental Research (UFZ), highly polluted with alkanes and benzene, toluene, ethyl-
Permoserstr. 15,
04318 Leipzig, Germany benzene, and xylene (BTEX) compounds. One of the in situ
bioremediation technologies directed toward volatile hydro-
M. Brennerova carbons, mainly BTEX and gasoline relying on the aerobic
Institute of Microbiology (IMIC), Czech Academy of Sciences, stimulation of the catabolic capabilities of the autochtho-
Videnska 1083,
142 20 Prague 4-Krc, Czech Republic nous bacteria, is air sparging (Marley et al. 1992; Reddy et
al. 1995; Bass et al. 2000; Hall et al. 2000; Heron et al.
D. H. Pieper : H. Junca 2002; Yang et al. 2005). However, despite the wide
Biodegradation Research Group, application of this technique to enhance the bioremediation
Helmholtz Centre for Infection Research (HZI),
Inhoffenstrasse 7, of nonchlorinated aromatic contamination in situ, there is
38124 Braunschweig, Germany still a scarcity of knowledge on the biocatalysts being
2. Appl Microbiol Biotechnol
stimulated and the overall microbiological characteristics of site. Several principal source zones of petroleum pollution
the process. In contrast, bioremediation studies often used were identified at the site which has a size of 28.3 ha—
to be restricted to follow the disappearance of hazardous three storage areas and the jet-fuelling depot. The pollutants
pollutants (Frostegard et al. 1993a; Frostegard et al. 1996) migrated significant down-gradient distances due to more
and still regard this system as a black box. As it is known than 20 years of massive fuel leakages in source areas and
that traditional culture-dependent methods are highly biased high permeability of sandstones. The amount of total petrol
when analyzing environmental samples (Amann et al. hydrocarbon (TPH) released into soil and groundwater until
1995), culture-independent methods have been applied 1997 is estimated as 7,150 t. At the start of the treatment,
since two decades in order to characterize microbial light nonaqueous-phase liquid (LNAPL) phase was fre-
community structures and their shifts under changing quently present in the wells with a thickness >0.5 m. The
environmental conditions. Lipid biomarker-based techni- pollution consisted mainly of jet fuel (70%) with admixture
ques (Guckert et al. 1991; White 1993; Frostegard et al. of gasoline and diesel.
1996; White et al. 1996; Zelles 1997; MacNaughton et al. Figure 1 shows a scheme of the Hradčany site and the
1999) provide culture-independent insights into several clean-up procedure carried out since 1997, when in situ
important characteristics of microbial communities such as technologies have been gradually applied. LNAPL soil
viable biomass, community structure, nutritional status, or vapour extraction (SVE) and air sparging (AS) with
physiological stress responses of the bacteria (Guckert et al. application of nutrient solutions (N, P, and K) have been
1991; Heipieper et al. 1996; Pennanen et al. 1996; applied to the site (Masak et al. 2003). The first clean-up
MacNaughton et al. 1999). However, the insight gained phase focused on maximum removal of LNAPL by vacuum
from lipid biomarker analysis primarily concerns nutritional extraction, whereas the second phase aimed at creating
or physiological status with little differentiation among favorable conditions enabling aerobic degradation in the
bacterial species. Complementary genetic methods targeting entire contaminated profile by AS and SVE. In the time
and discerning the sequence complexity of 16S rRNA frame of 1997–2006, 3,667 t of TPH were removed from
genes as a bacterial taxonomical biomarker allow the the site and it was estimated that biodegradation accounts
monitoring of taxonomical shifts in microbial community for 93%, vacuum extraction of LNAPL for 5%, and SVE/
structure at greater details (Janssen 2006). AS for 2% of the TPH amounts eliminated (Machackova et
The present study shows the monitoring of a former air al. 2005). In this study, the development of microbial
force base in the Czech Republic highly contaminated with communities in samples taken from three locations of that
jet fuel that is currently under bioremediation by the air site representing different stages of the treatment progress
sparging technique (Bass et al. 2000; Hall et al. 2000). The was studied using microbial community analyses by
site is a part of the Bohemian Cretaceous Basin, the most phospholipid fatty acid (PLFA) profiling and 16S rRNA
important resource of high-quality groundwater in the gene library analysis.
Czech Republic (Masak et al. 2003; Machackova et al. The results presented in this polyphasic approach show
2005). The endangered aquifer is the only source of links between the depletion of contaminants (in this case, a
drinking water in the region and the presence of extensive strong selector) in natural setups due to oxygen amendment
contamination limits future use and revitalization of the and an increase of the abundance and complexity of the
Fig. 1 Schematic representation
of the clean-up procedure
carried out since 1997 at
Hradčany site (AS air sparging,
VE venting, GWT ground
water table)
3. Appl Microbiol Biotechnol
autochthonous microbial soil community. Most probably, the previously lyophilized for 24 h and was carried out using
observed changes in the microbial community are related accelerated solvent extraction in an ASE 200 apparatus
and associated with the successful remediation of the soil. (Dionex), allowing an efficient extraction of lipids from
soils under high temperatures and pressures. Methanol,
chloroform, and buffer were applied in ratios described by
Materials and methods Bligh and Dyer (1959).
For the extraction, from each of the samples, 30 g of soil
Sampling were lyophilized and filled in an extraction cell (volume=
22 mL) together with the mix of solvents, heated for 5 min,
All samples were taken from a site of high kerosene and pressurized to 120 bar. The temperature and pressure
contamination, located in the Czech Republic, referred here were kept constant for 10 min (static extraction, two
as the Hradčany site. Since the Second World War until cycles). The total amount of solvent used for each cell
1990, the site was used as a military airport, and the was about 25 mL. The extracts were collected and
military activities resulted in an extensive contamination of separated by addition of appropriate volumes of distilled
the soil and groundwater by petroleum products (mainly by water and chloroform. The chloroform phase, which
jet fuel). The upper layers of the site (0.5–3 m) are formed contained the total fatty acids, was isolated and dried over
by quaternary river sediments (sands, gravels); the aquifer anhydrous sodium sulfate. The PLFA fraction was separat-
is composed of middle- to fine-grained Middle Turonian ed by liquid chromatography using silica gel columns
sandstone with a thickness of 67 to 75 m. The base of the (Bakerbond spe, Baker). By subsequent elution with
aquifer consists of Lower Turonian siltstones and marlites chloroform, acetone, and methanol, neutral glycolipids
with a thickness of about 75 m. The groundwater table and phospholipids were collected separately according to
depth varies from 3 to 8 m below the surface. In 1997, a Zelles (1997). The methanol fraction containing the PLFA
full-scale clean-up was initiated (Masak et al. 2003; was transesterified to the respective fatty acid methyl esters
Machackova et al. 2005). Soil samples were taken using (FAMEs) with trimethylchlorosilane in methanol (1:9, v/v)
spiral auger drilling technique. The actual level of the at 60°C for 2 h. The solvent was evaporated under a gentle
groundwater table (GWT) was measured prior to drilling in stream of nitrogen, and residues were resuspended in
the adjacent monitoring point for preliminary setting of hexane.
sampling depth. The three sampling sites are located within
the Hradcãny area (approximately 30 ha) with a reasonable Analysis of fatty acid composition by GC-MS and GC-FID
distance of several hundred meters between each other
(HRB-3: highest contamination, beginning of clean-up; Analysis of FAMEs in hexane was performed using a
HRB-2: 2.5 years of treatment; HRB-1: 5 years of quadruple GC system (HP8690, Hewlett & Packard, Palo
treatment). Samples were taken in the depth of 0.5 m Alto, USA) equipped with a split/splitless injector. A BPX-
above–1 m under the actual GWT level from the 0.2-m 5 capillary column (SGE, Darmstadt Germany; length,
layer of maximum contamination. All samples represent 30 m; inner diameter, 0.32 mm; 0.25 μm film) was used
very similar soils, both from the geological (soil scientific) for separation where the injector temperature was held at
and hydrogeological aspects. From each of the three sites, 240°C. The injection was splitless and He was used as
approximately 2 kg of soil were taken. The soil of each site carrier gas at a flow of 2 mL/min. The temperature program
was then homogenized in a sterile bucket and then packed was: 40°C, 2 min isothermal; 4°C/min to 230°C; 5 min
into glass jars and stored at 4°C under aerobic conditions. isothermal at 230°C. The pressure was held constant at
Sampling for petroleum hydrocarbon quantification was 7,57 psi. Additionally, a GC system with flame ionization
performed prior to soil mixing as petroleum contamination detector was used (Agilent 6890N) with a special FAME
quickly volatilizes during homogenization. Two split column (CP-Sil88 Varian Chromopack; length, 50 m; inner
samples for contamination content analyses were taken diameter, 0.25 mm; 0.2 μL film) to reach better separation.
from the sampled interval. Content of TPH was measured The pressure program was as follows: start, 27,64 psi for
by standard gas chromatography and infrared detection 2 min; increase, 0.82 psi/min up to 45.7 psi; isobaric for
(ISO TR 11046 and ISO TR11046[2]); BTEX was analyzed 5.5 min. The temperature program started at 40°C (2 min),
by standard gas chromatographic methods (EPA 601). increased 8°C/min up to 220°C, and was held there for
5 min. Injector temperature was 240°C, detector temperature
Fatty acid extraction and separation 270°C.
The peak areas of the carboxylic acids in total ion
The extraction of total fatty acids was performed with the chromatograms (TIC) were used to determine their relative
soil samples (five split samples of each site) that were amounts. The fatty acids were identified by their mass
4. Appl Microbiol Biotechnol
spectra and retention time compared to coinjections of (synthesized by Invitrogen, Karlsruhe, Germany). For
authentic reference compounds obtained from Supelco thermal cycling, a Hybaid PCR Express Thermocycler
(Bellefonte, USA). (Thermo Electron, Waltham, MA, USA) was used as
follows: initial denaturation at 94°C for 4 min, 35 cycles
Statistical analysis of 95°C for 45 s, 55°C for 45 s, and 72°C for 1.5 min.
These cycles were followed by one elongation step at 72°C
Principal component analyses (PCA) were applied on the for 7 min. PCR products were purified by using the
basis of numerical data matrices converted using the QIAquick PCR purification kit (Qiagen, Hilden, Germany)
program R (R: Copyright 2005, The R Foundation for and cloned in pGEM-T easy vector system (Promega,
Statistical Computing Version 2.1.1). The relative amounts Madison, WI, USA). Plasmid inserts were amplified by
of PLFA data were subjected to PCA to investigate the PCR with vector-specific M13 forward and reverse primers
interrelationships between the soil samples and to deter- (Sambrook et al. 1989) on transformant colonies dissolved
mine the predominant PLFA species in the samples. In the in water and previously incubated at 95°C for 10 min.
first attempt, the investigated soil samples corresponded to Amplified ribosomal DNA restriction analysis (ARDRA)
the object represented in the multidimensional space and was performed as previously described (Junca and Pieper
the PLFA values to the descriptors of the multivariate 2004). The purified PCR products were used as DNA
analysis. In a second PCA, reciprocal analysis was carried templates in independent sequencing reactions of both
out with the soil samples corresponding to the descriptors strands using the BigDye terminator v1.1 cycle sequencing
of the analysis. kit (Applied Biosystems, Foster City, CA, USA) using M13
primers and primers annealing at four different conserved
DNA extraction regions in two directions inside the 16S rRNA gene
sequences as described previously (Lane 1991). Sequencing
For DNA extraction, fractions of the soil samples were reactions were analyzed in an Applied Biosystems 3130xl
frozen with solid carbon dioxide at the time of sampling Genetic Analyzer (Applied Biosystems, Foster City, CA,
and maintained in this condition during transportation. USA) and sequence contigs were assembled using
Later on, samples were stored at −70°C until further Sequencher version 4.0.5 (Genes Codes, Ann Arbor, MI,
processing. DNA was extracted with the FastDNA Spin USA). The sequences were cleaned of vector sequences
kit for soil (QBiogene, Carlsbad, CA, USA) from 800 mg using VecScreen Blast program (NCBI, USA) and oriented
of soil per reaction tube, according to the instructions of the in 5′–3′ of the 16S rRNA genes using OrientationChecker
manufacturer with the only exception that the final elution (Bioinformatics Toolkit, Cardiff School of Biosciences,
of DNA from the filter was with 75 μL of Tris–HCl buffer UK). Sequences were analyzed for potential chimeric
3.33 mM pH 8.0. Five DNA extractions, equivalent to 4 g sequences with the service available at the Ribosomal
of soil, were performed for each soil sample, and extracted Database Project II (Cole et al. 2003). Additional potential
DNA were pooled together in a single reaction tube. The chimeras were assessed with the program MALLARD
DNA was dried and the final volume adjusted to 40 μL (Ashelford et al. 2006). The final datasets were aligned
with MilliQ water. DNA concentrations were quantified with the multiple sequence alignment method MUSCLE
using the Quant-iT PicoGreen dsDNA quantitation kit (Edgar 2004). A block of sequence alignments was selected
(Invitrogen—Molecular Probes Europe BV, Leiden, The with GeneDoc multiple sequence alignment editor software
Netherlands). (Nicolas 1997). A collection of the nearest neighbors to the
sequences obtained against the 16S rRNA gene sequences
PCR amplification, cloning, sequencing, and analyses reported and classified in the Ribosomal Database Project II
were found using Seqmatch (Cole et al. 2003). Neighbor-
The pooled DNA extracts were used as template in joining trees were calculated from the composite align-
polymerase chain reaction (PCR) amplifications with ments together with calculated bootstrapped values of 1,000
primers targeting two highly conserved regions identified trials using the functions implemented inside Clustal W
on bacterial 16S rRNA genes (Marchesi et al. 1998) [63F: (Thompson et al. 1994). Tree files were graphically
5′-CAG GCC TAA CAC ATG CAA GTC-3′ and 1387R: displayed with MEGA 3.1 software (Kumar et al. 2004).
5′-GGG CGG WGT GTA CAA GGC-3′]. The final For calculation of rarefaction curves and Shannon diversity
amounts or concentrations of the reagents for PCR in a indexes, the program DOTUR was used (Schloss and
volume of 50 μL were: 1X colorless GoTaq reaction buffer Handelsman 2005) using the distance matrices computed
(Promega, Madison, WI, USA), 5 U of GoTaq polymerase with Dnadist program (Felsenstein 1989) from the nucleo-
(Promega, Madison, WI, USA), 200 μM of dNTPs (MBI tide sequence alignments of the sequence libraries obtained
Fermentas, Germany), and 10 pmol of each primer in this study.
5. Appl Microbiol Biotechnol
Microbiological culture techniques from HRB-3 were even lower and only detectable after
PCR amplification. Thus, compatible results were obtained
Fractions of the soil samples were kept at 4°C after when comparing, as biomarkers, total DNA extract con-
collection. Colony forming units were determined in R2A centrations, CFUs, or PLFA concentration, which all point
agar (Difco, Livonia, MI, USA) in triplicates after plating to an increase in living microbial biomass.
of the appropriate dilution that were carried out on
phosphate buffer (50 mM, pH 7.0). Phospholipid fatty acid composition of Hradčany soils
The PLFA composition of Hradcãny samples (Fig. 2b) showed
Results significant differences depending on the time of air sparging
treatment. In the untreated samples, saturated fatty acids
Biomass development in the Hradčany soil (16:0, 18:0) are predominant next to 18:1Δ9cis fatty acid.
The major difference in the PLFA profiles between the three
Soil samples from the Hradčany site representative for investigated soil sampling sites was the significantly higher
different steps of the air sparging bioremediation process relative abundance of 16:1Δ9cis and 18:1Δ11cis monoun-
were investigated for pollutant content. In general, the three saturated fatty acids as well as cyclo19:0 cyclopropane fatty
sites can be characterized as follows: HRB-3, soil from an acid in the treated compared to the untreated samples.
untreated site contains high organic contamination (concen- A PCA of the PLFA profiles underlined the results given
trations of TPH=6,400 mg/kg and BTEX=4,400 mg/kg dry above. The first PCA (Fig. 3a) allowed to clearly
weight); HRB-2, soil from a 3-year clean-up site exhibits a distinguish PLFA patterns associated with the soil from
moderate organic contamination (TPH=3,900 mg/kg and three sites differing in the level of BTEX and kerosene
BTEX=190 mg/kg dry weight); and HRB-1, soil from a contamination and treatment duration. The data of all the
5.5-year clean-up site contains low organic contamination three different sampling sites formed distinct clusters. This
(TPH=1,500 mg/kg and BTEX=9 mg/kg dry weight). From PCA showed a clear separation of the three conditions on
each site, five samples were investigated which were taken in the biplot of the first two principal components, emphasiz-
the same drilling campaign. As the geological and hydro- ing changes in the PLFA composition of the soils according
geological specificities of all three sites were similar, the to the length of treatment. A separation of the soil samples
differences in microbiota are most likely due to the from the lowest level of contamination to the highest one is
difference in pollution level and cannot be explained by operating along the first principal component.
geological or other aspects. The second PCA stresses the dominant PLFAs that are
In order to compare the abundance and complexity of associated with the difference in the analyzed soil samples
microbial biomass of the samples subject to different times (Fig. 3b). The amount of variation explained by the first
of air sparging treatment, the overall abundance of PLFA and second principal components reached 86.3% of the
was analyzed (Fig. 2a). On the other hand, in the nontreated total variation. This PCA relates the abundance of specific
soil, which contains very high toxic concentrations of PLFAs (16:0; 18:0; 18:1cisΔ9) with the level of contam-
BTEX compounds, PLFAs were only present in very low ination (prior clean-up, HRB-3). On the other hand, other
amounts; this content increased by more than two orders of PLFAs (18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase in
magnitude in the air sparging treated soils. As PLFA are response to the treatment time and were particularly
only present in living (micro)organisms (MacNaughton et associated with the 3 years treated (HRB-2) samples. The
al. 1999; Kindler et al. 2006), this is a clear indication that other PLFAs were found in a close vicinity of the origin of
this bioremediation treatment leads to a significant increase the PCA plot, indicating that the relative amounts of these
in overall microbial biomass. The increase in biomass was PLFAs were not substantially affected by the level of
also reflected in strong differences between the soil samples contamination and the length of treatment.
regarding quantities of heterotrophic bacteria as quantified
by the number of colony forming units per gram of soil Molecular biological analysis of the microbiota
(CFU/g) (Fig. 2a) with HRB-3 exhibiting CFUs/g two composition of Hradčany soil
orders of magnitude lower than HRB-1. Analysis of DNA
concentrations by fluorescence quantification (see the To correlate shifts in lipid composition occurring at the
“Materials and methods” section) revealed a concentration investigated site with changes in bacterial taxonomical
of 40 ng dsDNA per gram of HRB-1 soil, whereas DNA complexity, the microbial community structures of the three
from HRB-2 was observable after gel electrophoresis but sampling points were assessed by 16S rRNA gene libraries.
below the concentration of 0.5 ng/μL dsDNA which could PCR clone libraries of 16S rRNA gene were generated from
be accurately quantified. Amounts of DNA extractable total pooled DNA extracts of the soils and initial screenings
6. Appl Microbiol Biotechnol
Fig. 2 Effect of air sparging
treatment on PLFA abundance
a 1.E+07 1.E+08
and composition in Hradčany
soils. a Biomass development,
Overall abundance of PLFAs (mA)
given as the overall abundance 1.E+07
of PLFA (filled diamonds, rep-
resented by area counts) and 1.E+06
CFUs (cell g-1)
colony forming units (open 1.E+06
squares) in the Hradčany site
caused by the air sparging treat-
ment. b PLFA patterns of soils
from the Hradčany site. No 1.E+05
treatment (HRB-3), 1.E+05
total BTEX concentration=
17,000 mg/kg; 3 years treatment 1.E+04
(HRB-2), total BTEX concen-
tration=960 mg/kg; 5.5 years
treatment (HRB-1), total BTEX
concentration=70 mg/kg. From 1.E+04 1.E+03
0 1 2 3 4 5 6
each sampling point, five
independent soil samples were Years of air sparging treatment
extracted and analyzed for their b 50
PLFA content. Standard devia-
tion of these five independent 45
measurements are given as
error bars 40
35
PLFA (%)
30
25
20
15
10
5
0
:0
:0
:1 :0
:0
ns 0
19 :0
:0
o
o
17 is
0
0
1
:0
16 s
:1 d 9
:1 1
n
cl
cl
:
5:
6:
d1
d1
c
14
15
16
17
18
19
20
15
tra
cy
cy
i1
i1
18 i s
:1
s
c
a
ci
tr a
16
18
:1
18
5.5 Years Treatment 3 Years Treatment No Treatment
performed by ARDRA. For HRB-3, ARDRA screening supplementary material). A global alignment of the
with AluI on 96 clones showed identical patterns in 82 of sequences obtained together with the most closely related
the clones, suggesting the predominance of a single 16S rRNA gene sequences from type strains and selected
taxonomic group in the library. A similar ARDRA sequences retrieved from public databases indicated the
screening on HRB-1 and HRB-2 clone libraries did not presence of sequences related to diverse evolutionary
give evidence for any predominant pattern. Further screen- branches (Janssen 2006). The clones in the clone libraries
ing by random sequencing was performed on 79 clones were assigned to operational taxonomic units (OTUs) using
from HRB-1, 80 clones from HRB-2, and 28 clones from >99% (OTU0.01), >95% (OTU0.05), and >90% (OTU0.1)
HRB-3. The relationships of 187 assembled sequences, sequence identity as criteria, as sequences with greater than
comprising a common 1-kb length block covering variable those identities are typically excluding differences based on
regions V2 to V6 of the 16S rRNA genes (Neefs et al. operon heterogeneity or are typically assigned to the same
1993), corresponding to positions 103 to 1130 of Escher- genus or order, respectively (Acinas et al. 2004b; Schloss
ichia coli 16S rRNA gene (GenBank accession number and Handelsman 2004).
J01695), are shown in Fig. 4a (expanded view and detailed Rarefaction analysis on each sequence library showed
labeling of these results are available as Electronic that the higher number of clones sequenced from the HRB-
7. Appl Microbiol Biotechnol
Fig. 3 PCA analyses of PLFA
a cyclo 19:0
0.4
patterns obtained from 18:1cis∆11
Hradčany soils. a Ordination
plot representing the relationship
between the contamination sites
and the PLFA patterns. The
19:0
cross indicates the origin of HRB-2
0.0
coordinates and values on the
5
cyclo17:016:0
i16:0
axes indicate the percentage of
16:1trans 14:0
the total explained variation. b GGG a15:0
G 16:1cis 17:0 18:0
PCA showing loading values for 20:0 18:1cis∆9
18:1trans 18:1cis
individual PLFA. PLFAs found
i15:0 15:0
on the right in the plot had
-0.4
PC 1 (26.6%) 16:0
increased in the no treatment
-0.4 0.0 0.4
(HRB-3) soils, whereas the ones
found in the lower part of the L
0
L L
plot had increased the 5.5-year L
II
Iu
u
treatment (HRB-1) and 3-year u II L
treatment (HRB-2) soils
HRB-1 HRB-3
-5
-
-4 -2 0 2
PC 1 (41.6%)
-4 -2 0 2 4
b
4
0.4
2
18:1cis∆9 18:0
20:015:0
PC 2 (34.3%)
cyclo19:0
itrans
16:0
14:0
18:1trans
i 15:0
cyclo17:0 HRB-3
0.0
a15:0
0
17:0
16:0
16:1cis
-2
-0.4
cyclo19:0
HRB-1
HRB-2
-4
18:1cis∆11
- 0 .4 0.0 0 .4
PC 1 (52.0%)
9. Appl Microbiol Biotechnol
ƒFig. 4 Taxonomical distribution of the 16S rRNA gene sequences Discussion
retrieved from the contaminated soils DNA under different bioreme-
diation treatments. a Neighbor-joining tree based on 16S rRNA gene
sequences obtained from HRB soil DNA amplifications. Circles The air sparging treatment of the Hradcãny site caused a
indicate sequences obtained by random screening of PCR clone significant increase in the amount of biomass and, at least
libraries of amplifications from DNA extracts of HRB-1 (blue), HRB- partially as a consequence, a decrease in organic contam-
2 (green), and HRB-3 (orange) soils. Light purple trapezoids indicate
ination of the soil. The increase in living microbial biomass
sequences of closely related bacterial type strains or cultured strains.
In cases where sequences with a similarity higher than 60% to an could be shown by us; compatible results were obtained
observed HRB-derived sequence were not available from bacterial when comparing, as biomarkers, total DNA extract con-
type strains, sequences from uncultured bacteria were included for centrations, CFUs, or PLFA concentration, which all point
orientation (violet triangles). Rarefaction curves for different distance
levels (OTU0.01, OTU0.05, and OTU0.10) for each of the analyzed
to an increase in living microbial biomass.
libraries were calculated by DOTUR (Schloss and Handelsman 2005) The major difference in the PLFA profiles between the
and are given below the dendrogram. Coverages (C) at 95% distances three investigated soil sampling sites was the significantly
were calculated according to Turing’s formula (Good 1953) where C= higher relative abundance of 16:1Δ9cis and specifically
100 represents complete coverage. b Relative clone frequencies in
18:1Δ11cis monounsaturated fatty acids in the treated
major phylogenetic groups (Order–Class) of the clone libraries
analyzed. HRB-derived 16S rRNA gene sequences were assigned to compared to the untreated samples. As cis-vaccenic acid
bacterial classes using the RDP-naïve Bayesian classifier according to (18:1Δ11cis) is synthesized via the so-called anaerobic
the taxonomical hierarchy of Garrity and Lilburn (release 6.0) with the pathway of fatty acid synthesis that is exclusively present in
default confidence threshold of 80%. The colors used in the stack
several Gram-negative bacteria (Keweloh and Heipieper
column diagram correspond to bacterial Orders as defined to the right
of the columns. Orders were grouped as Classes as shown to the very 1996), this indicates the high abundance of Gram-negative
right of the figure, except for Bacillales and Clostridiales that, for bacteria in the treated samples. The predominance of Gram-
simplicity, were grouped in the higher rank (phylum) of Firmicutes. negative bacteria in the treated samples is further supported
Sequences that could not be classified and that were retrieved only in
very low amounts were collectively indicated as “Unclassified
by the high abundance of Gram-negative-specific cyclo-
ribosomal genes.” For additional details of the sequences obtained propane fatty acids (cy17:0 and cy19:0) and the low
and control sequences used in this figure, see the accompanying abundance of Gram-positive-specific iso- and anteiso-
Electronic supplementary material branched fatty acids (i15:0, a15:0, i16:0, and i17:0).
However, inspection of discriminatory fatty acids shows
1 and HRB-2 libraries were indeed necessary to obtain the presence of Gram-negative-specific cyclopropane fatty
coverage comparable to the one for the HRB-3 library (see acids accounting for approximately 5% up to 40% of the
Fig. 4). total fatty acids, whereas abundance of Gram-positive-
Almost all the sequences obtained from HRB-3 were specific acids was negligible. This indicates also the
tightly clustering (>95% overall sequence similarity) inside untreated site to be dominated by Gram-negative organ-
the genus Pseudomonas with the majority of these sequences isms, and treatment to exert a significant effect on the
closely related, but not identical (identities >1,022/1,030, composition of the Gram-negative microbial community
99%), to those found in Pseudomonas cedrina or Pseudo- fraction. In fact, taking the combined abundance of the
monas azotoformans type strains inside the Pseudomonas Gram-positive-specific fatty acids and the Gram-negative-
fluorescens group (Anzai et al. 2000). Such clusters of specific fatty acids as a measure of the ratio between Gram-
sequence microdiversity in ribosomal genes are commonly positive and Gram-negative bacteria (Margesin et al. 2007),
observed in amplifications of environmental samples (Acinas the relative abundance of Gram-positive organisms was
et al. 2004a); however, its interpretation and significance is highest in the 5.5 years treated soil. The absence of
still under discussion. Nevertheless, it is very likely that, due polyunsaturated fatty acids shows that eukaryotes are
to a strong selection caused by the hazardous environmental practically absent in these soils, which indicates that a
conditions, only members of a bacterial genus tolerant to normal soil microflora has not been completely established
high solvent concentrations and possibly with the potential to by the so far carried out bioremediation process.
aerobically degrade such compounds were observed. HRB-2 The PCA carried out with data that stresses the dominant
exhibited a wider sequence diversification compared to the PLFAs associated with the difference in the analyzed soil
contaminated nontreated state (HRB-3). A distinct Pseudo- samples (Fig. 3b) clearly approves the tendencies visible
monas intragenus microdiversity was evidenced in this from the fatty acid profiles. The relation of specific PLFAs
sampling area with sequences highly similar to above- (16:0; 18:0; 18:1cisΔ9) with the highest level of contam-
mentioned P. cedrina/P. azotoformans cluster still being ination (prior clean-up, HRB-3) suggests that a highly
predominant (22% of clones) but 5% of the clone sequences specific microbiota is associated with these hazardous
being closely related (identities >1,016/1,030, 98%) to the environmental conditions. On the other hand, other PLFAs
recently described Pseudomonas rhizosphaerae type strain (18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase in response to
(Peix et al. 2003). the treatment time and were particularly associated with the
10. Appl Microbiol Biotechnol
3 years treatment (HRB-2) indicates a specific Gram- However, whereas there is only a small increase in the
negative bacterial community accumulating during the air Shannon diversity index from HRB-2 to HRB-1, both sites
sparging treatment. comprise very different microbial community compositions.
However, next to community shifts, changes in the Most importantly, Pseudomonas spp. was barely detectable
membrane fatty acid patterns of bacteria can also occur as (one out of 79 sequences) in HRB-1. In contrast, sequences
adaptive response to pollutant toxicity and environmental affiliated with Sphingomonadales, members of which had
stress conditions (Frostegard et al. 1993b; Heipieper and de been observed in various aromatic contaminated sites and
Bont 1994; Heipieper et al. 1996). Therefore, it is necessary which had been related to primary stages on polycyclic
to support the insights based on PLFA profiling also by other aromatic biodegradation (Leys et al. 2004, 2005), were
methods such as, e.g., molecular biological techniques. abundant only in HRB-1, comprising roughly 10% of the
Surprisingly, the expected trans–cis ratio of unsaturated fatty respective clone library, contrasting a single sequence in the
acids, a very useful parameter for stress monitoring in HRB-2 clone library.
bacterial cultures (Guckert et al. 1986; Guckert et al. 1991; In addition, Betaproteobacteria-affiliated sequences, a
Heipieper et al. 1992; Heipieper et al. 1996), did not show group only marginally detected (one sequence only) in
significant changes in the samples analyzed (data not HRB-2, are a significant fraction of the HRB-1 library,
shown), probably due to its transient identity. accounting for 20% of the total amount of sequences.
Although the PLFA analysis already demonstrated a shift Additional sequences exclusively observed in HRB-1
in the microbial community as well as an increase in living constitute a new branch inside the family Xanthomonada-
biomass, a detailed molecular biological analysis of the ceae (Gammaproteobacteria) with equal divergences (ap-
microbiota was necessary. Here, a clear increase in the proximately 15% of difference) against sequences from
microbial biodiversity of the site caused by the air sparging strains of the genera Frateuria and Rhodanobacter.
treatment was visible. Whereas almost all the sequences Bacterial assemblages similar to that of HRB-1 and
obtained from HRB-3 were clustering inside the genus consisting of Pseudomonas, Sphingomonas, Xanthomonas,
Pseudomonas, a tremendous increase in the identified Acidovorax, and Burkholderia sequences have been previ-
bacterial diversity occurred in the samples taken from 3 ously observed, for instance, at anthropogenic hydrocarbon-
and 5.5 years of treatment. contaminated coastal soils in Antarctica (Saul et al. 2005),
While Pseudomonas is a genus defined as ubiquitous while bacterial communities predominantly composed of
and of high environmental importance, these conclusions Pseudomonas, Sphingomonas, and Acidobacteria had been
are predominantly coming from observations using tradi- reported for instance in soil–groundwater ecosystems with
tional culture-dependent techniques (Moore et al. 2006) petroleum contamination (Popp et al. 2006).
which are generally accepted to include a severe bias As shown in Fig. 4b, a comparison of community
toward easy to culture microorganisms (Amann et al. composition at the level of bacterial classes, which is used
1995). However, our study and some other recent reports in many reports tracking shifts in microbial communities by
(Duineveld et al. 2001; Kaplan and Kitts 2004; Gerdes et al. FISH probes, T-RF sizes, or OTU definition (Pett-Ridge
2005; Popp et al. 2006; Ferguson et al. 2007) are showing and Firestone 2005; Yu et al. 2005; Watt et al. 2006; Allen
that Pseudomonas may be defined as a predominant et al. 2007; McGarvey et al. 2007) among others, would
member in communities of aerobic or microaerophilic direct to misleading conclusions as it would suggest the
ecosystems where high concentrations of crude oil are predominance and resilience of Gammaproteobacteria in
acting as a strong selector. the sites independent of the treatment. However, when
However, whereas HRB-3 sequences affiliated with comparisons are performed at the taxonomical scale Order,
Pseudomonas spp. comprise roughly 80% of all clones, only this shows to be an oversimplified assumption as strong
25% of HRB-2 clones were affiliated with that genus. Other shifts in composition inside the Order were observed. As an
predominant sequence types in HRB-2 were affiliated with example, among the Gammaproteobacterial sequences,
the classes of Actinobacteria, Acidobacteria, and Alphapro- only those affiliated with Pseudomonadales were predom-
teobacteria (predominantly members of the orders Rhizo- inant in the HRB-2 site but practically absent from HRB-1
biales and Rhodospirillales). The higher diversity observed where Xanthomonadales and Unclassified Gammaproteo-
in HRB-2 (Fig. 4) was also reflected by a higher Shannon bacteria are accounting for a relatively high amount of the
diversity index (H′, calculated for OTU0.05) of 2.67±0.25 total bacterial composition detected (>35%). This example
(95% confidence interval), compared to only 0.73±0.44 for shows how comparisons of taxonomical composition in
HRB-3. An even slightly higher value compared to HRB-2 bacterial communities should be at least at Order ranks.
was observed for HRB-1 (2.86±0.21), indicating diversity Lower resolution comparisons of Classes or even Phyla can
and balance of community composition to increase with be misleading and fail to detect significant community
bioremediation treatment time. changes.
11. Appl Microbiol Biotechnol
Integrating the information of overall microbial abun- necessarily able to degrade aromatics, but capable of
dance (Fig. 2a) with the sequences obtained (Fig. 4), it is surviving and growing in the cross-feeding mesh of
evident that even though the relative abundance of metabolites excreted from the initial biomass of degraders.
Pseudomonadales decreased in HRB-2 compared to HRB- In accordance with decreased concentrations of aromatics
3, the total number of Pseudomonadales cells per gram (and thus lower solvent stress), a higher variety of bacterial
of soil in HRB-2 is very likely by at least one order of taxonomical types and higher biomass content was ob-
magnitude higher. Thus, the initial predominance of served. This biodiversity restoration, which can be seen as
Pseudomonadales in HRB-3 may indicate a physiological an ecological succession, probably would not lead to the
advantage. These cells not only survive under these harsh same microbial composition of the soil as it was before the
conditions of low oxygen, high loads of aromatic carbon aromatic contamination occurred (Curtis et al. 2002).
pollutants, and high solvent concentrations, but obviously Here, it is shown that the bacterial community under
have been replicating under these conditions. This is in adaptation in these soils, concomitantly with the observed
accordance with culture-dependent studies on Pseudomo- degradation of the contaminants in situ, showed a dynamic
nadales (Heipieper and de Bont 1994; Sikkema et al. 1995; succession of Gram-negative bacteria with the community
Heipieper et al. 1996), which have shown members of this being initially restricted to Pseudomonadales at very low
group to be capable to replicate under harsh laboratory densities, developing an increased diversity comprising new
conditions and high solvent stress. It can thus be proposed proteobacterial types and Gram-positive bacteria. Compatible
that, at least at the site under study, Pseudomonads paved trends were observed using ordination methods, which
the road for other bacteria to be capable to replicate as showed the clear separation of the different fatty acid clusters
shown by the increased diversity observed in HRB-2. Thus, and indicated the predominance of Gram-negative bacteria
for a certain time, Pseudomonas is sharing its habitat in a able to resist the solvent concentrations at untreated
bacterial community of increasing complexity, as less contaminated soils and the diversification in samples where
restricting conditions for other phylotypes are being the treatment is applied. Future studies will focus on
generated during the clean-up process, leading to the catabolic activities in these sites and the relationship with
decrease in community predominance of Pseudomonas the phylogenetic changes observed by means of culture-
and the increase of other, previously not detectable dependent and culture-independent studies.
phylotypes. Particularly interesting is the increase in Acid-
obacterial sequence types, which are supposed to be Acknowledgments This work was supported by contract no.
003998 (GOCE) of the European Commission within its Sixth
selected in low-nutrient soil or in soil with a high amount Framework Program project BIOTOOL. We would like to thank the
of recalcitrant substrates (Torsvik and Ovreas 2002). As, excellent technical assistance of Silke Kahl.
moreover, soils with a high content of nutrients showed
positive selection for Alphaproteobacteria and specifically
Gammaproteobacteria (Amann et al. 1995), the ratio
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