Africa-US Network of Centers of Excellence in Water & Environmental Science & Technol....

Africa-US Network of Centers of Excellence in Africa-US Network of Centers of Excellence in Water & Environmental Science & Technol.Water & Environmental Science & Technol.

Bioremediation of Soils in AfrricaBioremediation of Soils in AfrricaRamble O. Ankumah. Ph.D.Ramble O. Ankumah. Ph.D.

Tuskegee UniversityTuskegee University

Overview of Soil As Media For Overview of Soil As Media For Biological & Chemical ReactionsBiological & Chemical Reactions 1. Soils contain solids and fluids 2. The solids are various inorganic and organic

compounds 3. The fluids are liquids (water being the

solvent) and gases 4. The volume of the components of agricultural

soils at optimum soil moisture for plants is approximately as follows:

Field View of SoilField View of Soil

a. Soil vs. Regolith Regolith: Unconsolidated material above

bedrock: Quite universal» 1. Can be negligible (shallow) or hundreds of

feet thick» 2. May be material weathered from underlying

rock or might have been transported by H2O or wind and deposited upon the bedrock or upon other material covering bedrock.

Soil Colloids and Their Soil Colloids and Their Importance in Soil FunctionImportance in Soil Function

Colloidal Materials in SoilsColloidal Materials in Soils

» I. Clay Minerals

» II. Soil organic Colloid (Humus)

Clay MineralsClay Minerals

Types of Clay MineralsTypes of Clay Minerals

» 1. Layer Silicates

» 2. Hydrous oxides of Iron and aluminum

» 3. Amorphous Clays

Oxides, Hydroxides and Oxides, Hydroxides and OxyhydroxidesOxyhydroxides

Hydrous Oxides of Iron and AluminumHydrous Oxides of Iron and Aluminum» pH Dependent and variable» Amphoteric i.e. can have positive or

negative charges» Positive charge under low pH» Negative charge under high pH (0-

4mmolc/100g)

Soil Colloids: Organic ColloidsSoil Colloids: Organic Colloids

Organic ColloidsOrganic Colloids» pH Dependent and variable» Structure has Carboxylic(–COOH),

Phenolic, and hydroxyl (-OH) groups. » Dissociation of this groups at various pH

results in negative charge.» Charge always negative (150 and 550

cmolc/kg)

Carbon Cycle on Soil Organic Carbon Cycle on Soil Organic Matter (SOM)Matter (SOM)

A. Organic Constitution of Plants:A. Organic Constitution of Plants: Divided into six broad categories1. Cellulose -most abundant 15-60 %2. Hemicellulose 10-30%3. Lignin 5-30%4. Water soluble fraction

(simple sugars, amino acids, and aliphatic acids) 5-30%

Carbon Cycle: Carbon Cycle: Assimilation/MineralizationAssimilation/Mineralization

5. Ether and alcohol soluble (Waxes, fats oils

resins and a number of pigments) 6. Protein (have N and S) 7. Mineral Component-usually

estimated by ashing 1-13%» As plant ages the content of water soluble

fraction decreases and the percentage of cellulose, hemicelluose and lignin rises.

B.B. Carbon AssimilationCarbon Assimilation 1. Organic Matter serves two functions a. Provides energy b. Supplies C for new cell materials

» Products: CO2, CH4, organic acids, Alcohol

Assimilation: Process of converting substrate to protoplasmic carbon

» Most microbial cells contain 50% » Under aerobic conditions 20-40% of substrate C is

assimilated and remainder released as CO2.

» Fungal flora more efficient in C transformation than other groups and releases less CO2.

» Fungi and actinomycetes more efficient than Bacteria.

» During Decomposition Fungi 30-40% C metabolized is used to form new mycelium.

» Actinomycetes 15-30%» Aerobic Bacteria less efficient, assimilates

to 5-10%» Anaerobic Bacteria 2-5% assimilated

Immobilization: Assimilation of inorganic substrates into complex organic molecules.

» Determined by utilization of nutrients elements for cell synthesis.

» Magnitude is proportional to net microbial cells or filament formed and is related to C assimilated by a factor governed by C:N, C:P, C:K, or C:S ratio of the newly generated protoplasm.

C. C. Decomposition and CODecomposition and CO22 Evolution: Evolution:

» Most important function of microbial flora is decomposition of organic matter and release of CO2.

» Degradation is a property of organotrophs (heterotrophs).

D.D. Process in Organic Matter Process in Organic Matter Transformation. Transformation.

» 1. Plant and animal constituent disappear under influence of microbial enzyme.

» 2. New microbial cells are synthesized.» 3. Certain end products of the breakdown

are excreted to the surroundings.

E.E. Decomposition of Soil Organic MatterDecomposition of Soil Organic Matter» Rate of CO2 release during mineralization

of humus varies with soil type » Factors governing humus decomposition

are» a. Organic Matter Level in soil» b. Cultivation

» c. Temperature

» d. pH

» e. Depth

» f. Aeration

Fresh substrates sometimes accelerate and sometimes reduce humus decomposition.

Enhancement is known as priming. 2-5% C present in humus can be

mineralized per annum. Cultivation enhances O. M. Decomposition.

E.E. Breakdown of Added Carbonaceous Breakdown of Added Carbonaceous Materials:Materials:

» Factors affecting breakdown of added organic materials.

» a. Type of material» b. Temperature» c. O2

» d. pH» e. Inorganic nutrients» f. C/N ratio of plant tissue

Nitrogen is the key substrate for microbial growth and hence organic matter breakdown.

If the N content is high and the substrate is easily metabolized, organism satisfies N needs from this source.

If substrate is poor in N, decomposition is slow, and C mineralizes will be stimulated by the supplement.

F.F. Mineralization of the Materials and Mineralization of the Materials and C:N C:N Ratio.Ratio.

» 1. During mineralization of the compounds containing little N, the C:N ratio tends to decrease with time.

» 2. This results in gaseous loss of C while N remains in organic combinations for as long as C:N ratio in wide.

» 3. Thus % of N in residue continues to rise as decomposition progresses.

» 4. Curve approach approximately ratio of 10:1.

» 5. C:N ratio of soil is roughly 10:1» 6. Microbial cells have C:N ratio

between 5 to 15 parts C to 1 parts N. approximately 10:1.

Arrangement of plants in order of decreasing rate of Mineralization.

1. Sweet Clover 3.14%2. Alfalfa 3.07%3. Red Clover 2.20%4. Soybeans 1.85%5. Millet 1.17%6. Flax 1.73%7. Corn Stalks 1.20%8. Sudan Grass 1.06%9. Wheat 0.5%10.Oat Straw 0.61%

Low N content or a wide C:N ratio is associated with slow decay.

Example: Incorporation of a residue having a wide C:N ratio.

» 1. Microbes will develop to the extent of available N and other inorganic

nutrients.» 2. All immediately available N will be

Polysacharides: Cellulose: Polysacharides: Cellulose: IntroductionIntroduction

Insoluble polymer of β(1-4) linked glucose The most abundant organic carbon material

in nature Molecular weight appox. 20,000-2.4 million Most bugs hydrolyze α-linkages Because it occurs with other materials and

is resistant to degradation it impedes enzymatic attack

Cellulose: IntroductionCellulose: Introduction

Generally animals don't metabolize it. Older plant tissues have a lot of it. Cotton fibres about 90% cellulose. About 14,000 isolates can degrade it. Bugs degrading it need supply of N. Decomposition faster under aerobic

conditions when N-fertilizers are applied.

Factors Affecting Cellulose Factors Affecting Cellulose DegradationDegradation

Occurs from freezing to 65 C, Slow in frozen state Thermomophiles 45-65 optimum Mesophiles 25-35 C, tolerate 15-54 C Psychrophiles < 20 C Most soil microbes are mesophiles Cellulose not available as source of energy Slow type of release, conversion to glucose is very

Microbiology of Cellulose Microbiology of Cellulose DegradationDegradation

Microbes growing on it don't grow fast on cellulose.

Ability to degrade cellulose is common in fungi but unusual with bacteria and actinomycetes.

Fungi Degrading Cellulose:» Aspergillus, Fusarium, Chaetomium, Phoma

Bacteria:» Most popular is Cytophaga, Pseudomonas spp.

Cellulose Degradation: MicrobiologyCellulose Degradation: Microbiology

Actinomycetes:» e.g. Streptomyces*, Norcodia*, Micromonospora.

* Very versitile All cellulose degrading bacteria and

actinomycetes can be isolated at pH 5.5. Montmorillonite reduces activity of cellulases. Cellulose exploited in industry by breaking and

fermenting it to ethanol

HemicelluloseHemicellulose

Chemistry: Not a polymer of just glucose but a mixture

of glucose, galactose, mannose, glucoronic acid, galactoronic acid, arabinose, and xylose.

Hemicellose, generally 50 to 200 units. Polysacharide may exist as a linear chain as

with cellulose. Usually branched.

Degradation of HemicelluloseDegradation of Hemicellulose

Only few sugars and uronic acids are common. These are:

Pentoses (5 C sugars)-Xylose and Arabinose;

Hexoses (6 C sugars)- Mannose, glucose, and galactose;

Uronic acids-glucouronic acid and galactouronic acids.

II.II. Degradation of HemicelluloseDegradation of Hemicellulose

a. Easily degraded than cellulose; b. Hemicellulose disappears initially

at a rapid rate and may be due to degradation of part which

is physically available. c. Converted to CO2 and microbial

cells.

Degradation of Hemicellulose: FactorsDegradation of Hemicellulose: Factors

2. Factors Affecting Decomposition

» a. pH approximately 7

» b. O2 limiting

» c. Age Older plant more resistant to decomposition.

» d. Availability of inorganic nutrients

e.g. N

Microbiology of Microbiology of Hemicellulose DegradationHemicellulose Degradation

More microbes can degrade it; Microbes not specific, can degrade other things. Microorganisms Utilizing Hemicelloluse: 1. Bacteria

Organism SubstrateBacillus Mannan, galactomannan, xylan

Cytophaga Glactan Erwinia XylanPseudomonas XylanStreptomyces Mannan, Xylan

III. Microbiology of III. Microbiology of Hemicellulose DegradationHemicellulose Degradation

2. Fungi

Organism Substrate

Alternaria Arabinoxylan, xylan

Aspergillus Araban, arabinoxylan, mannan.

ChaetomiumArabinoxylan

Fusarium Araban, arabinoxylan

Penicillium Araban, mannan

Lignin: IntroductionLignin: Introduction

a. Lignin is the third most abundant plant tissue» Most common in woody plants, i.e. about

15-35%.» they are slowly degraded. » Woody plants contribute most of the

lignin which are degraded by the microflora.

Lignin : I. IntroductionLignin : I. Introduction

b. Lignin is chemically complex and is found in cell walls and also in the middle lamella.

c. Lignin can also be found in certain fungi and algae

d. Not much known about the biochemistry of lignin degradation.

Lignin: DecompositionLignin: Decomposition

a. Very resistant to degradation (enzymatic)

b. Occurs in the presence of oxygen. c. Rate observed far less than cellulose,

hemicellulose, and other carbohydrates. d. Well decomposed material has high

percentage of lignin.

Lignin DecompositionLignin Decomposition

e. Aerobic decomposition of corn stalks. » 2/3 of total dry matter lost in 6 months and 1/3

of lignin in 6 months. f. Lignins of young tissues disappear more

rapidly than mature plants. g. Lignin protects the decomposition of

associated polysaccharides by mechanically separating the microorganisms from CHO.

Factors Affecting Rate and Extent of Factors Affecting Rate and Extent of Lignin Decomposition.Lignin Decomposition.

1. Temperature» Optimum temperature is 30oC» No degradation less than 7oC or greater than

37oC. 2. Availability of nitrogen 3. Aeration 4. Plant residue undergoing decay

» Methoxy groups first-more under anaerobiosis and least under aerobic conditions

V. Microbiology of Lignin DegradationV. Microbiology of Lignin Degradation

Numbers of microbes capable of breaking down lignin are small.

1. Major microflora are Fungi» Decomposition of lignin is primarily by fungi.» color of the decayed substrate is indicative of the

mode of attack.

Microbiology of Lignin DegradationMicrobiology of Lignin Degradation

» i. White-rot-Fungi: » White rot-fungi are the most active lignin-

degrading microorganisms.

» Their degradation leads to CO2 and H2O

» Species involved are mainly basidiomycetes (Phanerochaete sp., most studied) and a few ascomycetes.

Microbiology of Lignin DegradationMicrobiology of Lignin Degradation

» a. Basidiomycetes-Agaricus, Armillaria, Fomes, Pleurotus, Coriolus etc.

» b. Ascommycetes: Xylaria, Libertella and Hypoxylon

» White rot fungi are thought to degrade lignin only in the presence of other degradable substrates which they use as primary energy source

» ii. Brown-rot Fungi.» These degrade the polysaccharides associated

with lignin and remove the CH3 subgroups, and R-O-CH3 side chains.

» This leaves the phenol behind which turn brown upon oxidation.

» Representative groups include Poria and Gloeophyllum

» iii. Soft-rot Fungi.» Important in wet situations and appear to

degrade hardwood lignin more effectively than soft woods.

» Representative groups are Chaetomium and Preussia

2. Aerobic bacteria e.g. Arthrobacter, Pseudomomas, Xanthomonas,

Flavobacterium, and Micrococcus.

3. Actinomycetes have been implicated e.g. Streptomyces and Norcardia.

Actinomycetes and Bacteria such as Streptomyces and Norcardia and anaerobic G- bacteria such as Azotobacter and Pseudomonas , depolymerize the lignin structure and lower the molecular size.

VI. Biochemistry of Lignin Degradation:VI. Biochemistry of Lignin Degradation:

4. To obtain energy and carbon from the transformation, the ring is opened and the cleavage products either enter the metabolic pathway or are used in biosynthesis.

5. Ring opening proceeds by 3 ways.» A. Ortho fission

» B. Meta Ring Fission

» C Gentisic Acid Pathway

VI. Biochemistry of Lignin Degradation:VI. Biochemistry of Lignin Degradation:

1. All 3 pathways require molecule O2.

2. Reason not much aromatic degradation in anaerobic environment.

3. Oxygenase involved is mainly dioxygenase i.e. in ortho Fission.

4. Meta Fission limited to alkyl substituted rings.

Soil Organic Matter (SOM)Soil Organic Matter (SOM)

Degradation of Plant Residues» Mineralization of less resistant C

compounds leads to CO2

» Less resistant C compounds persist

» Rate of decomposition influenced by C/N ratio of material

Organic Matter EquilibriumOrganic Matter Equilibrium

C = bm/kWhere: C= The % of soil organic carbon in

Equilibrium (tons/ha) b= The annual amount of fresh organic

matter added (tons/ha) m= Conversion rate of fresh organic matter

in to soil carbon (percent).

Organic Matter EquilibriumOrganic Matter Equilibrium

k= the annual decomposition rate of the soil organic carbon (percent)

a= the annual addition of organic matter (tons/ha)a = bm

b valuesb values

Temperate Forest = 3-15 tons/ha

Tropical Forest = 1-8 tons/ha

Temperate Praires = 1.5 tons/ha

Tropical Savannas = 0.5- 1.5 tons/ha

k and m valuesk and m values

k is primarily a function of temperature

» Values range from 2-5 % in tropical forest to about 1.5 in tropical savannahs.

» Temperate forest values range from 0.4-1%

m is about 30-50% per year

Organic Matter EffectOrganic Matter Effect

1. Direct physical/Chemical- soil structure and chemical properties

2. Direct biological Effect- Mineralization/Immobilization

3. Indirect Biological Effect- Alter/Modify physical and chemical processes

Organic Matter ManagementOrganic Matter Management

1. Manipulate existing organic matter through tillage or soil drainage

2. Crop residue placement on or in soil

and burning to enhance management operations

Organic Matter Mgt.Organic Matter Mgt.

3. Augment in situ production using green manure, cover or sod crops

4. Amendment with organic matter sources such as animal waste and or compost

In Situ Production of In Situ Production of Organic CropsOrganic Crops

1. Legume crops

2. Green crops

3. Animal manure

Use of Enzyme Activity in Use of Enzyme Activity in Evaluation BioremediationEvaluation Bioremediation

Evaluation of the use of soil enzyme activities and microbial diversity in determining agricultural and environmental impacts of “soil quality”.- Tillage Practices: Conventional vs

Conservation- Organic Farming

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Bioremediation of Contaminated Bioremediation of Contaminated Soil-ApproachesSoil-Approaches

Bioremediation strategy uses microbes, plants or microbial or plant enzymes to detoxify contaminants.

» Biodegradation

» Mineralization

» Cometabolism

Criteria for BioremediationCriteria for Bioremediation

Organisms must have catabolic activity to degrade contaminant at a reasonable rate

Target contaminant must be bioavailable Site must have soil conditions conducive to

microbial and plant growth

Bioremediation StrategiesBioremediation Strategies

Passive or intrinsic bioremediation Biostimulation Bioventing Bioaugmentation Landfarming Composting Phytoremediation

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