(5)Industrial and Environmental Uses of Recombinant Microorganisms

Bioremediation and Biomass

• Bioremediation: Using biological agents to remove toxic substances.

• Biomass: Waste from food/agricultural industries, now valuable.

• Importance: Environmental cleanup and commercial applications.

Microbial Degradation of Xenobiotics

• Toxic waste disposal is a major environmental issue.

• Soil microbes can degrade herbicides, pesticides, solvents, etc.

• Pseudomonas species are key in breaking down over 100 organic compounds.

• Degradation requires enzymes from one or more pathways.

• Genes can be on chromosomal DNA, plasmids, or both.

• Xenobiotics convert to catechol and protocatechuate, then acetyl-CoA, succinate, pyruvate, or acetaldehyde.

Halogenated Compounds Degradation

• Degradation is inversely proportional to the number of halogen atoms (F, Cl, Br, I).

• Dehalogenation: replacement of halogen with a hydroxyl group via nonselective dioxygenase.

• Dehalogenation can occur before or after biodegradation.

Genetic Engineering of Biodegradative Pathways

• Limitations of Bioremediation:

  • No single microbe degrades all wastes.

  • High compound concentrations inhibit microbes.

  • Contaminated sites have chemical mixtures, causing inhibition.

  • Non-polar compounds adsorb onto soil, reducing availability.

  • Microbial degradation can be slow.

Workarounds for Genetic Engineering

• Conjugation: Transfer plasmids with different pathways into a recipient microbe.

• Fusion plasmids: Recombination of two plasmids with homologous DNA.

• Co-existing plasmids: Multiple plasmids in one bacterium (if compatible).

  • Results in single bacterium having multiple capabilities

Organophosphate Pesticides

• Detoxification methods: chemical treatment, incineration, or landfill.

• Pseudomonas diminuta MG and Flavobacterium spp. have organophosphorus hydrolase for pesticide hydrolysis.

• Degradation is slow due to limited compound uptake by bacteria.

Cell Surface-Expressed Enzymes

• E. coli engineered to produce organophosphorus hydrolase on the cell surface.

  • Enhanced activity and stability.

  • Limited to lab scale.

Utilization of Biomass - Starch and Sugars

• Starch: biopolymer with linear (amylose) and branched (amylopectin) homopolymers.

  • Amylose: linear chains of 100- 400,000 D-glucose residues linked by α-1,4-bonds

  • Amylopectin: short linear chains of approx. 17 to 23 glucose units, linked by α-1,4-bonds and joined by α-1,6 -linkages and some 1,3-linkages to form a highly branched structure that contains 10,000-40,000,000 glucose residues

Commercial Production of Fructose and Alcohol

• Step 1: Gelatinization via steam cooking.

• Step 2: Liquefaction with a-amylase at 50-60°C to hydrolyze α-1,4-linkages.

• Step 3: Saccharification with glucoamylase for complete hydrolysis.

Improving Alcohol Production

• Overproduce enzymes in fast-growing recombinant microorganisms.

• Use heat-resistant α-amylase (80-90°C).

• Alter α-amylase & glucoamylase genes for same optimum pH and temperatures to combine liquefaction and saccharification.

• Develop enzymes for raw starch degradation.

• Employ fermentation organisms to synthesize/secrete glucoamylase.

Utilization of Cellulose

• Lignocelluloses classes:

  • Primary cellulosics: plants for cellulosic content (cotton, timber, hay).

  • Agricultural waste cellulosics: post-harvest materials (rice hulls, corn stovers, straw).

  • Municipal waste cellulosics: waste paper, cardboards.

Lignin

• 3D, globular, irregular, insoluble polymer (>10,000 MW).

• Phenyl propane subunits with irregular chains.

• Bonds are hard to hydrolyze.

Hemicellulose

• Short-chain, heterogenous polymers with hexoses and pentoses.

  • Xylans: hardwood

  • Glucomannans: Softwoods

• Three major types:

  • Xylans: backbone of poly-β-1,4-xylan with side links of glucuronic acid, arabinose and arabinoglucuronic acid

  • Mannans: composed of glucomannans, galactomannans

  • Arabinogalactans

Cellulose

• Abundant polymer with long chains of D-glucose in β-1,4 configuration.

• Hydrolyzable to glucose.

• Crystalline structure, insoluble and resistant to hydrolysis.

Cellulase

• Requires breakdown into structural components via acid, base, high temperature/pressure.

• Bacteria/fungi degrade using cellulase (multiprotein complex).

• Component enzymes:

  • Endoglucanase

  • Exoglucanase

  • Cellobiohydrolase

  • β-glucosidase/cellobiase

Genetic Engineering Strategies for cellulose

• Clone bank of DNA from a cellulolytic prokaryote

• Uses for cloned cellulase genes

  • The cellulose binding domain coding gene of the cellulase complex can be fused to a commercial protein encoding gene

  • Cellulase genes have been introduced directly into the S.cerevisiae and Z.mobilis

Biobutanol/Hydrogen Production

• Butanol vs. Ethanol:

  • Higher energy content (110,000 vs 84,000 Btu/gallon)

  • Less evaporative than ethanol and gasoline.

  • Can be shipped via existing pipelines.

  • Usable as gasoline replacement (100%).

ABE Fermentation

• Bacterial fermentation to produce acetone, butanol, and ethanol from starch.

• Anaerobic process using Clostridia (e.g., Clostridium acetobutylicum).

• Produces solvents in a 3:6:1 ratio (acetone:butanol:ethanol).

Hydrogen Production

• Bacterial fermentation of glucose yields limited hydrogen (2-3 mol/mol vs. potential 12 mol/mol).

• Microbial fuel cells (MFCs) generate bioelectricity from cellulose via electrogenesis.

• Exoelectrogenic bacteria transfer electrons to the MFC anode.

Microbial Electrolysis Cell

• Add a voltage to MFC and omit oxygen to produce hydrogen.

• With bacterial voltage generation (0.3V), add 0.2V to generate hydrogen.

• Process extends biohydrogen production past fermentation dead-end products (e.g., acetic acid).

Bacterial Species and Xenobiotic Breakdown

• Pseudomonas species are key in breaking down over 100 organic compounds because they possess versatile metabolic pathways and enzymes capable of degrading a wide range of xenobiotics.

Endpoint Products of Xenobiotic Degradation and Effect of Halogenation

• Xenobiotic degradation often results in simpler, less toxic products like CO2, H2O, and inorganic salts.

• Halogenation generally increases the resistance of xenobiotics to biodegradation because the halogen atoms increase the stability of the compound and make it less susceptible to enzymatic attack.

Limitations and Solutions in Microbial Bioremediation

• Limitations of Microbial-Based Bioremediation:

  • Limited bioavailability: Pollutants may be adsorbed to soil or present in low concentrations.

  • Microbial specificity: A single microorganism may not be able to degrade the entire range of pollutants.

  • Environmental conditions: Suboptimal temperature, pH, or nutrient availability can limit microbial activity.

  • Toxicity: High concentrations of pollutants can be toxic to microorganisms.

• Ways to Overcome Limitations:

  • Bioaugmentation: Adding specific microorganisms to enhance degradation.

  • Biostimulation: Optimizing environmental conditions (e.g., nutrients, oxygen) to stimulate native microbial activity.

  • Cometabolism: Introducing a primary substrate that supports the degradation of a secondary pollutant.

  • Genetic engineering: Modifying microorganisms to enhance their degradative capabilities.

Mesophilic vs. Psychrophilic Microorganisms

• Mesophiles: Grow best at moderate temperatures (20-45°C). Their enzymes function optimally at these temperatures.

• Psychrophiles: Grow best at low temperatures (below 20°C). Their enzymes are adapted to function in cold environments.

• Effect on Bioremediation: Mesophiles are more effective in warmer climates, while psychrophiles are better suited for cold environments.

Superbug (Pseudomonas putida)

• The first patented bacterium (Superbug, Pseudomonas putida) was created by Ananda Chakrabarty and was used to degrade crude oil. It was developed by combining plasmids from different Pseudomonas strains, each capable of degrading different hydrocarbons.

Bioremediation of PCBs

• The current approach for bioremediation of PCBs involves a combination of anaerobic and aerobic degradation. Anaerobic bacteria can dechlorinate PCBs, while aerobic bacteria can further degrade the dechlorinated products. Often involves bioaugmentation and biostimulation.

Genetically Modified Microorganisms for Organophosphate Pesticide Degradation

• Pseudomonas, Flavobacterium, and E. coli have been genetically modified to express organophosphorus hydrolase (OPH), which degrades organophosphate pesticides like parathion and glyphosate.

Composition of Starch (Amylose vs. Amylopectin)

• Amylose: A linear polymer of glucose linked by α-1,4-glycosidic bonds.

• Amylopectin: A branched polymer of glucose with α-1,4-glycosidic bonds in the linear chain and α-1,6-glycosidic bonds at the branch points.

Production of Ethanol and Fructose from Starch

• Ethanol: Starch is hydrolyzed to glucose, which is then fermented by yeast (e.g., Saccharomyces cerevisiae) to produce ethanol.

  • Biotechnology Improvements: Genetic engineering of yeast to improve ethanol tolerance, increase substrate utilization, and reduce byproduct formation.

• Fructose: Starch can be converted to fructose-rich syrups using enzymes like glucose isomerase.

  • Biotechnology Improvements: Improved enzyme production and optimization of isomerization process.

Composition of Lignocellulose

• Lignin: A complex polymer of phenylpropanoid units that provides structural support.

• Cellulose: A linear polymer of glucose linked by β-1,4-glycosidic bonds.

• Hemicellulose: A branched polymer of various sugars (e.g., xylose, mannose, galactose) linked by different glycosidic bonds.

• Least Useable for Ethanol Production: Lignin is the least usable because it is difficult to break down and not readily fermentable to ethanol.

Cellulase and Cellulosome

• Cellulase: A group of enzymes that hydrolyze cellulose into glucose.

• Cellulosome: A multi-enzyme complex produced by some microorganisms that efficiently degrades cellulose.

  • Composition:

  • Scaffoldin: A structural protein that binds the cellulases.

  • Cellulases: Including endoglucanases, exoglucanases, and β-glucosidases.

• Enzymatic Biodegradation of Cellulose:

  • Endoglucanases: Cleave internal β-1,4-glycosidic bonds randomly, creating free chain ends.

  • Exoglucanases (cellobiohydrolases): Cleave cellobiose (a disaccharide) from the ends of cellulose chains.

  • β-glucosidases: Hydrolyze cellobiose into glucose.

Isolation of Cellulase Genes

• Prokaryotes: Metagenomic sequencing of environmental samples, followed by functional screening.

• Eukaryotes: cDNA library construction from cellulase-producing fungi, followed by expression cloning.

Ethanol vs. Butanol as Fuels

• Ethanol:

  • Production: Traditional yeast fermentation of sugars.

  • Advantages: Well-established production process.

  • Disadvantages: Lower energy density, can be corrosive.

• Butanol:

  • Production: ABE (acetone-butanol-ethanol) fermentation by Clostridium species.

  • Advantages: Higher energy density, less corrosive.

  • Disadvantages: (Not specified in the provided reference.)

Hydrogen Production

• Bacterial fermentation of glucose yields limited hydrogen (2-3 mol/mol vs. potential 12 mol/mol).

• Microbial fuel cells (MFCs) generate bioelectricity from cellulose via electrogenesis.

• Exoelectrogenic bacteria transfer electrons to the MFC anode.

• Microbial Electrolysis Cell

  • Add a voltage to MFC and omit oxygen to produce hydrogen.

1. Endpoint Products of Xenobiotic Degradation and Effect of Halogenation

   • Xenobiotic degradation often results in simpler, less toxic products like CO2, H2O, and inorganic salts.

   • Halogenation generally increases the resistance of xenobiotics to biodegradation because the halogen atoms increase the stability of the compound and make it less susceptible to enzymatic attack.

2. Limitations and Solutions in Microbial Bioremediation

   • Limitations of Microbial-Based Bioremediation:

     • Limited bioavailability: Pollutants may be adsorbed to soil or present in low concentrations.

     • Microbial specificity: A single microorganism may not be able to degrade the entire range of pollutants.

     • Environmental conditions: Suboptimal temperature, pH, or nutrient availability can limit microbial activity.

     • Toxicity: High concentrations of pollutants can be toxic to microorganisms.

   • Ways to Overcome Limitations:

     • Bioaugmentation: Adding specific microorganisms to enhance degradation.

     • Biostimulation: Optimizing environmental conditions (e.g., nutrients, oxygen) to stimulate native microbial activity.

     • Cometabolism: Introducing a primary substrate that supports the degradation of a secondary pollutant.

     • Genetic engineering: Modifying microorganisms to enhance their degradative capabilities.

3. Mesophilic vs. Psychrophilic Microorganisms

   • Mesophiles: Grow best at moderate temperatures (20-45°C). Their enzymes function optimally at these temperatures.

   • Psychrophiles: Grow best at low temperatures (below 20°C). Their enzymes are adapted to function in cold environments.

   • Effect on Bioremediation: Mesophiles are more effective in warmer climates, while psychrophiles are better suited for cold environments.

4. Superbug (Pseudomonas putida)

   • The first patented bacterium (Superbug, Pseudomonas putida) was created by Ananda Chakrabarty and was used to degrade crude oil. It was developed by combining plasmids from different Pseudomonas strains, each capable of degrading different hydrocarbons.

5. Bioremediation of PCBs

   • The current approach for bioremediation of PCBs involves a combination of anaerobic and aerobic degradation. Anaerobic bacteria can dechlorinate PCBs, while aerobic bacteria can further degrade the dechlorinated products. Often involves bioaugmentation and biostimulation.

6. Genetically Modified Microorganisms for Organophosphate Pesticide Degradation

   • Pseudomonas, Flavobacterium, and E. coli have been genetically modified to express organophosphorus hydrolase (OPH), which degrades organophosphate pesticides like parathion and glyphosate.

7. Composition of Starch (Amylose vs. Amylopectin)

   • Amylose: A linear polymer of glucose linked by α-1,4-glycosidic bonds.

   • Amylopectin: A branched polymer of glucose with α-1,4-glycosidic bonds in the linear chain and α-1,6-glycosidic bonds at the branch points.

8. Production of Ethanol and Fructose from Starch

   • Ethanol: Starch is hydrolyzed to glucose, which is then fermented by yeast (e.g., Saccharomyces cerevisiae) to produce ethanol.

     • Biotechnology Improvements: Genetic engineering of yeast to improve ethanol tolerance, increase substrate utilization, and reduce byproduct formation.

   • Fructose: Starch can be converted to fructose-rich syrups using enzymes like glucose isomerase.

     • Biotechnology Improvements: Improved enzyme production and optimization of isomerization process.

9. Composition of Lignocellulose

   • Lignin: A complex polymer of phenylpropanoid units that provides structural support.

   • Cellulose: A linear polymer of glucose linked by β-1,4-glycosidic bonds.

   • Hemicellulose: A branched polymer of various sugars (e.g., xylose, mannose, galactose) linked by different glycosidic bonds.

   • Least Useable for Ethanol Production: Lignin is the least usable because it is difficult to break down and not readily fermentable to ethanol.

10. Cellulase and Cellulosome

    • Cellulase: A group of enzymes that hydrolyze cellulose into glucose.

    • Cellulosome: A multi-enzyme complex produced by some microorganisms that efficiently degrades cellulose.

      • Composition:

        • Scaffoldin: A structural protein that binds the cellulases.

        • Cellulases: Including endoglucanases, exoglucanases, and β-glucosidases.

    • Enzymatic Biodegradation of Cellulose:

      • Endoglucanases: Cleave internal β-1,4-glycosidic bonds randomly, creating free chain ends.

      • Exoglucanases (cellobiohydrolases): Cleave cellobiose (a disaccharide) from the ends of cellulose chains.

      • β-glucosidases: Hydrolyze cellobiose into glucose.

11. Isolation of Cellulase Genes

    • Prokaryotes: Metagenomic sequencing of environmental samples, followed by functional screening.

    • Eukaryotes: cDNA library construction from cellulase-producing fungi, followed by expression cloning.

12. Ethanol vs. Butanol as Fuels

    • Ethanol:

      • Production: Traditional yeast fermentation of sugars.

      • Advantages: Well-established production process.

      • Disadvantages: Lower energy density, can be corrosive.

    • Butanol:

      • Production: ABE (acetone-butanol-ethanol) fermentation by Clostridium species.

      • Advantages: Higher energy density, less corrosive.

      • Disadvantages: (Not specified in the provided reference.)

13. Hydrogen Production

    • Bacterial fermentation of glucose yields limited hydrogen (2-3 mol/mol vs. potential 12 mol/mol).

    • Microbial fuel cells (MFCs) generate bioelectricity from cellulose via electrogenesis.

    • Exoelectrogenic bacteria transfer electrons to the MFC anode.

    • Microbial Electrolysis Cell

      • Add a voltage to MFC and omit oxygen to produce hydrogen.

• Bioremediation: Using biological agents to remove toxic substances.

• Biomass: Waste from food/agricultural industries, now valuable.

• Xenobiotic: A chemical substance found in an organism that is not naturally produced or expected to be present within the organism.

• Mesophilic Bacterium: A bacterium that grows best in moderate temperature ranges, typically between 20°C and 45°C.

• Psychrophilic Bacterium: A bacterium that thrives in cold temperatures, typically below 20°C.

• Superbug: A term referring to the first patented bacterium, Pseudomonas putida, created by Ananda Chakrabarty, used to degrade crude oil.

• Starch: biopolymer with linear (amylose) and branched (amylopectin) homopolymers.

• Amylose: linear chains of 100- 400,000 D-glucose residues linked by α-1,4-bonds

• Amylopectin: short linear chains of approx. 17 to 23 glucose units, linked by α-1,4-bonds and joined by α-1,6 -linkages and some 1,3-linkages to form a highly branched structure that contains 10,000-40,000,000 glucose residues

• Lignocellulose: plant dry matter (biomass) that is composed of lignin, cellulose and hemicellulose

• Lignin: 3D, globular, irregular, insoluble polymer (>10,000 MW).

• Hemicellulose: Short-chain, heterogenous polymers with hexoses and pentoses.

• Cellulose: Abundant polymer with long chains of D-glucose in β-1,4 configuration.

• Cellulase: Requires breakdown into structural components via acid, base, high temperature/pressure.

• Cellulosome: multi protein complex

• ABE Fermentation: Bacterial fermentation to produce acetone, butanol, and ethanol from starch