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10- Bioremediation and microorganisms

Microorganisms in Biotechnology

V. Bergougnoux – 7/12/2017

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xenobiotic (xenos - foreigner, bios - life) - a foreign artificial compound that is not created by natural processes

Difficult and slow decomposition (persistent), toxic for plants, animals and humans

Source of xenobiotic Examples

Agriculture Pesticides, herbicides

Health Synthetic therapeutics

Food Additives, flavors, dyes

Energetic industries CO2, SO2, particles, ashes

Transport NOx, CO2

Consumption industries Plastics, dyes, coatings

Accumulate in living organisms: the higher in the food chain the more contaminated and at risk you are getting heavier doses; the heavier the dose, the more lethal

Bio-accumulation: case of mercury

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Most xenobiotics: are lipophilic penetrate membranes by diffusion are transported by lipoproteins in blood require chemical conversion to facilitate excretion

In human, xenobiotic metabolism consist mainly into deactivation (mainly in liver) and excretion (breath, urines, sweat).

Two-phase process: Phase I reaction: a hydrophilic functional group (-OH, -NH2, -SH) is

introduced enzymatically - e.g. hydroxylation: monooxygenases of cytochrom P450

Phase II reaction: a hydrophilic group is added/conjugated.

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Bioremediation: reactions of natural attenuation, which includes all biotic and abiotic processes used to reduced contaminant contents in the environment.

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Biodegradation: primary mechanism to reduce biodegradable contaminants by employing organisms such as bacteria, fungi, algae and plants.

Transformation: result in partial or complete detoxification of a contaminant or can create a compound even more toxic than the initial one.

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Mineralization: the parent compound is completely degraded to CO2, new cell mass, and water. This is a highly desirable result for toxic contaminants.

Time to Biodegrade…

Paper towel 2-4 weeks

Newspaper 6 weeks

Apple core 2 months

Was coated milk carton 3 months

Cotton gloves 1-5 months

Wool gloves 1 year

Plywood 1-3 years

Painted wooden sticks 13 years

Plastic bags 10-20 years

Tin cans 50 years

Disposable diapers 50-100 years

Plastic bottle 100 years

Aluminium cans 200 years

Glass bottles undetermined

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Contaminant compounds are transformed by living organisms through reactions that take place as part of their metabolism

Carbon act as the source of cell building material and electrons as the source of energy

Microbes catalyzes the oxidation of the organic compounds (contaminants) that cause transfer of electron from organic chemicals to some electron acceptor

Aerobic organisms use oxygen as the final electron acceptor and organic carbon as C source

Anaerobic organisms use sulfate or carbon dioxide as the electron acceptor

Facultative organism utilize nitrates, iron and manganese as electron acceptor

Many chlorinated solvents degrade faster as electron acceptors. In these cases, an electron donor is added to the system to begin the process. As the substrate is metabolized under anaerobic conditions, an electron is released and then used to replace a chlorine atom on the chlorinated solvent.

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In-situ bioremediation: • Soil and associated ground water are treated in place without

excavation • Microbes responsible for the stabilization and treatment of waste

products containing organic matter • But they require a minimum of oxygen and nutrients

Ex-situ bioremediation: The soil is excavated prior to treatment

Ex-situ slurry: Creation and maintenance of soil-water slurry as

bioremediation medium

Bioventing: supply air and nutrients via wells, takes advantage of indigenous microorganisms

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Bioventing: supply air and nutrients via wells, takes advantage of indigenous microorganisms

In situ biodegradation: supply air and nutrients by circulating aqueous solutions through contaminated soils or groundwater

Bioventing: supply air and nutrients via wells, takes advantage of indigenous microorganisms

In situ biodegradation: supply air and nutrients by circulating aqueous solutions through contaminated soils or groundwater

Biosparging: injection of air below the water table, increasing the groundwater oxygen concentration and mixing in saturated zone

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Bioventing: supply air and nutrients via wells, takes advantage of indigenous microorganisms

In situ biodegradation: supply air and nutrients by circulating aqueous solutions through contaminated soils or groundwater

Biosparging: injection of air below the water table, increasing the groundwater oxygen concentration and mixing in saturated zone

Bioaugmentation: addition of indigenous and exogenous microorganisms; limits: competition and necessity

Biostimulation

(monitored) Natural attenuation: capacity of indigenous microbes without additional enhancement; relies on biological, physical and chemical processes. Feasible only when the biodegradation rate is higher than contamination

Land farming: • Simple process; well suited for shallow soil surface (10-35 cm of soil) • Contaminated soil is excavated and spread over a prepared ground; soil is

periodically tilled to improve aeration • Remediation due to indigenous microorganisms, as well as chemical and

physical processes • Includes organic wastes and problematic wastes • 1/2 –life for degradation of diesel fuel and heavy oil: 54 days

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Composting: • Cost-expensive, restricted to highly contaminated environments • Composting material (straw, bark and wood chips) is mixed with the

contaminated soil and piled into heaps • Temperature increases to 60°C and above, favoring the growth of

thermophilic bacteria • Temperature above 70°C achieved after 6-22 days of incubation; 84-86% of

contaminant was removed after 40 days (only 35% in untreated soil).

Biopiles: • Combination of land-farming and composting • Control physical losses of contaminants

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Bioreactors: Refers to any vessel or container where biological degradation of

contaminants is isolated and controlled 2 types of bioreactors: dry and slurry:

- Dry bioreactors: treatment only with microbes and nutrients. Adequate moisture for microbial growth is maintained by sprinkler system or rainfall. Aeration is done my physical mixing - Slurry bioreactors: wider range of pollutants. Soil is mixed in equal or greater amount of water. Microbes and nutrients are added to form a soil slurry. Conditions are easily maintain compared to dry reactors.

A function of: Microorganisms Environmental factors Contaminant type & state

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Aerobic bacteria: Shown to degrade pesticides and hydrocarbons; alkanes and

polyaromatics May be able to use the contaminant as sole source of carbon and

energy Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus and

Mycobacterium Methanotrophs:

Aerobic bacteria that utilize methane for carbon and energy Methane monooxygenase has a broad range of substrates

Anaerobic bacteria: Not used as frequently as aerobic bacteria Can often be applied to bioremediation of polychlorinated

biphenyls (PCBs) in river sediments, trichloroethylene (TCE), and chloroform

Fungi: Able to degrade a diverse range of persistent or toxic pollutants

the degradation of a wide range of organic compounds precedes a period in which their apparent destruction is not known - the period of adaptation (the acclimation period, the phase of adaptation, lag phase)

during this period, the substance may spread to surface or groundwater

this phase is longer under anaerobic conditions

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biological detoxification by microorganisms is strictly dependent on the concentration of the substance in the environment (high concentrations of toxic pollutant can suppress the growth of microorganisms)

the detoxification is usually accomplished by a simple primary reaction, most often by hydrolysis, hydroxylation, dehalogenation, demethylation or other dealkylation, methylation, reduction of the nitro group, deamination, ether cleavage, conversion of the nitrile to amide and conjugation

Co-metabolism microbial metabolism of an organic substance that can not be used as a source

of essential nutrients and energy one is the transformation of xenobiotics with an enzyme with very low

substrate specificity, which can catalyze also the conversion of substances structurally similar to the natural carbon source

Synergism the co-operation of several microorganisms to convert organic matter synergism involves microorganisms that can not separately transform or

degrade the pollutant synergistic responses are often responsible for growth factors, in particular

vitamins from group B and amino acids

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in order for the pollutant to be biodegradable, it must exist in a form that is available to organisms

it is usually necessary to dissolve in water

occurrence in non-aqueous liquid phase (NAPL) and sorption to soil material reduces its availability for bacterial cells

some changes in the chemical nature of the pollutant (complex formation, protonation, deprotonation) may make it less "suitable" for biodegradation

influenced by the ability to transfer the pollutant molecule through the cell membrane

microorganisms using NAPLs produce surfactants (emulsifiers or biosurfactants)

surfactants facilitate the release of the NAPL from the aqueous phase by producing small droplets of less than 0.1 μm

typical emulsions can also be formed in which the water immiscible droplets are so small that they last for an unlimited period of time in the aqueous phase

pseudosolubilization - the hydrophobic substrate released from NAPL is incorporated into the micelle center and in this form is fed to the aqueous phase

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production of surfactants or emulsifiers has been described mainly in microorganisms which can degrade NAPLs formed by alkanes

an important limiting factor for cell degradation activity is the size of the droplets

produced

more than 100 species of microorganisms are involved in processes of biological degradation of petroleum substances

in a petroleum-contaminated environment, the percentage of oil-depleting micro-organisms increases sharply (but no isolated micro-organism is able to degrade all components of oil)

most micro-organisms decomposing petroleum hydrocarbons produce emulsifiers

among the most important oil and oil degradatives belong Pseudomonas, Achromobacter, Arthrobacter, Acinetobacter, Flavobacterium, Brevibacterium, Corynebacterium, Nocardia, Candida, Rhodotorula, Sporobolomyces, Penicillium and Cunninghamella

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the biodegradation of petroleum hydrocarbons may take place over a very wide temperature range

at low temperatures, the rate of biodegradation can be limited by light volatile hydrocarbons which are toxic to a number of micro-organisms and, at higher temperatures, from the contaminated system quickly plow

lower temperatures are particularly suitable for the degradation of paraffin fraction of crude oil, the role of co-metabolism

nutrient enrichment is needed - the acceleration of the biodegradation process

in the marine environment, the ratio of C: N is most often considered a ratio of 10: 1, for C: P then 100: 1, the ratio between C: N is 60: 1 and C: P = 800: 1

anaerobic degradation - nitrate or sulfate as an electron acceptor

n-alkanes-monooxygenase or dioxygenase reactions monoterminal oxidation, the resulting fatty acid is further oxidized in the -oxidation

process; Pseudomonas oleovorans diterminal oxidation; Corynebacterium sp. subterritorial oxidation, formation of secondary alcohols and subsequently ketones

which are a substrate for monooxygenase reactions, the product is acetylester further hydrolyzed to alcohol and fatty acid which is subsequently oxidized in the β-oxidation process; Nocardia

highly branched isoprenoid alkanes (pristan - 2,6,10,14-tetramethylpentadecane) are mostly subjected to ditermine oxidation to form dicarboxylic acids; very slow and

is usually inhibited by terminal branching of the hydrocarbon; Brevibacterium erythrogenes, Corynebacterium sp., Nocardia globerula

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aromatic hydrocarbons (phenol, benzene, toluene, ethylbenzene, xylenes, styrene, atrazine) their degradability is often limited by various substituents (by chlorination and

methylation)

often requires the presence of another readily degradable compound, presence of electron acceptors

key are the enzymes dioxygenase (hydroxylation)

aerobic degradation - Pseudomonas putida, P. fluorescens, P. aeruginosa, Burkholderia cepacia, B. stearothermophilus, Cryptococcus elinovic

the splitting of the benzene nucleus also occurs during the anaerobic degradation of benzoic acid phototrophic bacterium Rhodopseudomonas palustris and two denitrifying species of Thauera aromatica and Azoarcus evansii

polyaromatic hydrocarbons (PAH) (phenanthrene, naphthalene, anthracene, benzo (a) pyrene) very resistant to microbial attack (low water solubility, high sorption ability) include compounds with two or more fused benzene rings which may be differently

substituted PAHs with two to three aromatic rings are better biodegradable than PAH with four or

more cycles the rate-limiting step for aerobic bacteria is the initial oxidation of the aromatic ring dibenzofuran - Sphingomonas sp .; phenanthrene - Streptomyces flavovirens, sea

cyanobacteria Agmenellim quadruplicatum and Rhodomonas baltica, fungi Phanerochaete chrysosporium

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halogen compounds (tetrachloromethane, trichloroethylene, DDT) halogenated alkanes and alkenes can be reductively dehalogenated in the anaerobic

environment, anaerobic microorganisms mostly replace the halogen atom with a hydrogen atom and, in the case of a higher degree of halogenation, the process of dehalogenation of the hydrocarbon can take place in several stages

some halogenated compounds undergo microbial hydrolytic dehalogenation during which halogen in an aromatic or other molecule is replaced by OH or with simultaneous dehydrohalogenation

Rhodococcus rhodochrous, Pseudomonas fluorescens, P. cepacia, P. putida, Methanosarcina spp., Xanthobacter autotrophicus, Alcaligenes sp.

unsaturated halogenated hydrocarbons are most commonly digested anaerobically (Dehalococcoides ethenogenes, Sporomusa ovata), but also aerobic degradation (Mythylosinus trichosporium, Burholderia cepacia, Pseudomonas putida)

DDT-1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane one of the oldest and best known insecticides (synthesis - 1874, insecticidal effects -

1939, Nobel prize - 1943)

there is no known microorganism that uses DDT as the only source of carbon and energy

there are various methods for the biological degradation of DDT aerobic and anaerobic

bacteria: Escherichia coli, Enterobacter aerogens, Enterobacter cloacae, Klebsiella pneumonia, Pseudomonas aeruginosa, Pseudomonas putida, Bacillus sp., Hydrogenomonas, Staphylococcus sp., Stenotrophomonas sp.

fungi: S.cervisiae, Phanerochaete chrysosporium, Trichoderma viridae, Aspergillus flavus, Mucor circinelloides, F. oxysporum

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polychlorinated biphenyls (PCBs) contain a biphenyl nucleus with varying degrees of chlorination

very persistent, strongly hydrophobic and therefore accumulate mainly in soil and in water sediments (mainly from 3 or more chlorine atoms)

have considerable resistance not only to microbial attack but also to heat and chemical decomposition

for certain microorganisms, biphenyl and monochlorobiphenyl are a growth substrate (although they grow better on conventional substrates)

in the sediments the anaerobic degradation of PCBs was described by so-called reductive dechlorination

anaerobic dechlorination was carried out by a methanogenic consortium

Acinetobacter sp., Alcaligenes sp., Achromobacter sp., Bacillus brevis, P. cruciviae, Klebsiella pneumoniae, A. niger, Shewanella oneidensis

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serves to remove pollutants which are dissolved or dispersed in waste water so that they are not capable of sedimentation

the principle is the biological culture of microorganisms which, as part of their life processes, acquires them as building materials and energy sources and is separable from the purified waste water by a simple physical process (usually sedimentation)

an important parameter is BOD5 (BOD) - serves for the indirect determination of organic substances that are subject to biochemical degradation under aerobic conditions; it is the amount of oxygen consumed by microorganisms at biochemical processes to decompose org. substances in water; a routine, worldwide standardized method is used - the so-called dilution method for determining a five-day BOD

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The following input parameters are required for biological wastewater treatment:

sufficient organic substances subject to aerobic biodegradation

for a long time adapted by microorganisms,

the absence of toxic substances,

sufficient dissolved oxygen,

pH without extreme values and without sudden changes,

temperature in the range of 5 to 35 ° C,

not the extreme concentrations of dissolved inorganic salts,

ratio between BOD5: N: P at least 100: 5: 1.

technological processes of aerobic biological treatment of sewage water in sewage plants are generally divided into technologies with biologically saturated surface-based biological culture (biological filters) and technologies with biological culture in the flow (activation tanks)

biofilters - tanks filled with piece material that is cracked mechanically by pre-treated sewage; after a certain time of incorporation, a slimy coating of microorganisms is created on the cartridge - the principle of purification is biological, therefore it is not true in the sense of filtration

biologically purified water is taken from biological filters to settling tanks with a residence time of about 1.5-2h (excess biological coating is flushed with purified water)

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Activation is one of the most commonly used methods of biological treatment of both urban and industrial waste water

the waste water is mixed with so-called activated sludge with sufficient aeration and mixing

activated sludge - suspended mixture of flake - forming, fibrous and loose micro - organisms in waste water and other solid organic and inorganic substances - flakes (zooglea) with a diameter of 0.1 mm

a typical dry sludge of activated sludge is in the range of 2-10 g / l

activated sludge flakes usually have the ability to sediment (necessary for successful biological purification!)

contains 5 · 109-1.5 · 1010 bacterial cells / ml, mainly: Acinetobacter and Zooglea ramigera (polysaccharide gels), Pseudomonas, Bacillus, Micrococcus, Alcaligenes, Moraxella, Flavobacterium; nitrifying bacteria - Nitrosomonas, Nitrobacter, Thiobacillus

Besides bacteria, higher organisms include, for example, vortices (Vorticella opercularia), protozoa, nematodes - consumers who live microorganisms from activated sludge and allow their disposal

the activated sludge is capable of removing a significant amount of organic contamination, as well as nitrogen and phosphorus compounds

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besides biochemical processes, sorption - adsorption of colloidal and some dissolved substances on flakes and sludge is also important

after a sufficiently long contact time of the wastewater with the activated sludge in the activation tank, the mixture is passed to the settling tank, where the activated sludge flakes are separated from the purified wastewater

during the activation of the amount of sludge is constantly increasing - by synthetic processes a new biomass is formed, the part of which, the so-called sludge, is removed from the settling tank outside the process for separate disposal and the part is returned (recirculated) back to the process

limiting the production of excess sludge using a membrane reactor - a microfiltration unit in the place of the settling tank

Anaerobic methods: they are mainly used for concentrated industrial waters or for the disposal of organic

sludge

the process is sometimes called anaerobic digestion and involves a complex interaction of three groups of bacteria:

1. fermentative / hydrolytic bacteria - facultative and obligatory anaerobic, produce exogenous hydrolyzing substances (proteins, lipids, polysaccharides) for simpler substances (acetic, butyric, propionic, CO2, H2, methanol); Clostridium (C. cellulosolvens, C. butyricum, C. thermocellum), Ruminococcus (R. flavefaciens), Acetivibrio (A. cellulolyticus), Bacteroides (B. cellulosolvens) - 108-109 bacteria in 1 ml of anaerobic sludge; most active at neutral pH

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2. acidogenic bacteria - Clostridium thermoaceticum, Acetobacterium woodii, Syntrophobacter, Syntrophomonas, Desulfovibrio - oxidize organic acids to H2, CO2 and acetic acid; are not too sensitive to changes in external conditions; optimal pH 5-6

3. methanogenic bacteria - Methanosarcina (M. berkeri, M. frisius), Methanobacterium (M. thermoautotrophicum, M. formicicum), Methanococcus, Methanomicrobium - strictly anaerobic, belongs to the Archa domain, terminating the conversion of previous products to methane; hydrotreatment (they form methane from H2 and CO2) and aceto-nitrophonic (form methane from acetic acid); extremely sensitive to temperature fluctuations and pH changes (optimum 6.5-7.2); require a higher concentration of trace elements (Ni, Co, Mo)

processes of acidogenesis and acetogenesis are strongly influenced by the concentration of hydrogen in the environment (higher concentrations reduce process speeds)

acetogenic and methanogenic bacteria are dependent on one another - syntrophy its metabolism is enhanced when the anaerobic sludge is in the form of dense granules,

which facilitates the transfer of H2 and other decomposition intermediates For example, the upstream anaerobic sludge blanket or EGSB (expanded granulated

sludge blanket)

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Advantages of anaerobic processes: methane production - most of the energy gain in anaerobic processes exceeds the energy

needed to maintain the necessary conditions (especially temperature); the acquired energy can be used for heating buildings or for electricity production and its use in the operation of the device (aeration blowers in aerobic processes) or sale

low power consumption (do not need to aerate) reducing the total sludge mass by converting the organic matter to CH4, CO2 and H2O;

usually 30-65% of the raw solids in the sludge are usually removed, which can significantly reduce the sludge disposal costs

removal or reduction of the sulfur content of the carbonate is released during combustion into the air in the form of oxides, where it is emitted by air humidity in the form of acid rain

sulfur is present in coal as inorganic (Fe disulfides, sulfides, sulfates, elemental sulfur) or organic

sulfur can be removed by bacterial leaching using a bacterium oxidizing sulfur compound - Thiobacillus ferroxidans (optimum temperature is 26-30 ° C and pH 1.8-2.0) or the thermoacidophilic archea Sulfolobus acidocaldarius

bacterial leaching reduces the sulfur content of black and brown coal

elimination of sulphide minerals from coal is not economically beneficial due to the energy requirements of the reaction system

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the principle of removing heavy metals, metalloids or radionuclides from the liquid source is the absorption by biomass of microorganisms (live and dead)

a very fast process that is not affected by metabolic inhibitors

it is mainly accomplished by complexing cell surface structures, ion exchange or microprecipitation

availability, mobility and bonding of metals affect physicochemical factors - mainly pH and temperature (preferably pH 4-8)

adsorption must be followed by desorption into the solution to further use the biomass - bicarbonate, EDTA, ammonium carbonate

biosorption methods are most commonly used to clean contaminated low-metal waters where it does not pay for the use of selective sorbents for the accumulation or isolation of a metal from the mixture

Organismus Sorbovaný kov

Rhizopus arrhizus Ag, Au, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Th, U, Zn

Saccharomyces cerevisiae Ag, Co, Cu, Th, U, Zn

Candida tropicalis Cd, Cr, Cu, Ni, Zn

Kluyveromyces marxianus U

Aspergillus niger Au, Cu, U

Penicillium chrysogenum Cd, Cr, Cu, Hg, Pb, U, Zn

Bacillus subtilis Au, Cd, Cu, Fe, Mn, Ni, Pb, Zn

Bacillus licheniformis Au, Cu, Fe, Mn, Ni

Citrobacter sp. Pb, U

Chlorella vulgaris Au, Cd, Ni, Pb, U, Zn

the most suitable sorption material is the biomass of fungi and yeasts mycelium fungi is grown in flow culture or surface fermentation "bed" (shelf) reactors are used where absorption takes place or the metals can be

removed by passage through the chitin column from the cell wall of the fungus

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it has been shown that cell surface modification can significantly affect the binding of metal ions, for example chemical: washing with detergents, crosslinking with organic solvents or acid or lye

during continuous industrial processes, the technique of immobilizing live or dead cells into matrices (eg insoluble Ca-alginate, polyacrylamide, etc.) can be used to remove metals from sewage - very stable systems that allow long-term use of accumulated biomass

(wood shavings, clay, sand, gravel, ZrO2) and a porous material (foam, polysulfide or other sponge), the use of microbial biofilms for the industrial removal of heavy metals from sewage -

intracellular accumulation is often irreversible, and drastic methods (ashing or dissolution in acid or lye) are used to release the metal,

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