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Biochemical Engineering II Presentation: Bioreactors & Kinetics
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ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 12, 2025 © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II About Me Ph.D. in Chemical Engineering, NC State University, USA (2024) Bryce Canyon National Park M.Sc. in Chemical Engineering, BUET (2019) B.Sc. in Chemical Engineering, BUET (2017) Research Field-deployable sensors for plant disease detection and plant health monitoring Upper Antelope Canyon Arches National Park Fun Fact Love traveling (explored more than 30 states) and listening to music. © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Learning in Biochemical I (ChE 473) ▪ The concept of biological catalysis. ▪ Microorganisms: Nature of microorganisms, their importance and classification; Industrially important microorganisms. ▪ Biomolecules: Amino acids and proteins; Metabolic stoichiometry and energetics; Molecular genetics and control systems. ▪ Kinetics of enzyme catalyzed reactions: Michaelis-Menten equation; Immobilized- enzyme technology; Enzyme-linked Immunosorbent Assay; Immobilized enzyme kinetics. Batch fermentation: Biomass yield and product formation; Rates of reaction; Growth; Limiting substrate concentrations; Monod’s equation. ▪ Introduction to food microbiology and environmental biotechnology. © 2025 by Oindrila Hossain, PhD What to Expect From Biochemical II (ChE 475) ▪ Advanced bioreactor design and operation ▪ Transport phenomena in bioprocesses (mass transfer, oxygen transfer) ▪ Downstream processing and product recovery ▪ Bioprocess scale-up and industrial applications ▪ Advancement in human and plant disease detection ▪ Understanding of recombinant DNA technology, cell signalling mechanisms, and ChE 475 Biochemical Engineering II imaging techniques in bioprocessing © 2025 by Oindrila Hossain, PhD Relative Scale of Biological Molecules ChE 475 Biochemical Engineering II How big are human cells? https://www.nature.com/scitable/content/the-relative-scale-of-biological-molecules-and-14704956/ © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Eukaryotes vs. Prokaryotes Eukaryotes Prokaryotes Nucleus present No true nucleus Multicellular Unicellular Larger (10– 100 μm) Typically smaller (0.1–5 μm), however, a much larger (centimeter-long) bacterium has recently been discovered in a mangrove swamp. https://www.technologynetworks.com/cell-science/articles/prokaryotes-vs-eukaryotes-what-are-the-key-differences-336095 © 2025 by Oindrila Hossain, ChE 475 Biochemical Engineering II Biomolecular Building Blocks Building Blocks Larger units © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Biomolecular Building Blocks (Cont’d) Amino Acids and Proteins https://www.jpt.com/support-contact/resources/amino-acids/ https://www.technologynetworks.com/applied-sciences/articles/essential-amino-acids-chart-abbreviations-and-structure-324357 Found in some archaea and bacteria, involved in methane-producing enzymes (Not universally recognized in all organisms). © 2025 by Oindrila Hossain, PhD Biomolecular Building Blocks (Cont’d) ChE 475 Biochemical Engineering II Antigen-Antibody Interaction Antibody structure Each antibody is highly specific to a particular antigen. https://www.biologyexams4u.com/2012/11/antigen-antibody-interaction.html Shete, Prajakta & Patil, Rakesh & Pawar, S.. (2015). Electrochemical nano-immunosensors for early detection of tumor markers. Annual Journal of University Research. A. J. Univ. R., 4 (2014) 225. © 2025 by Oindrila Hossain, PhD Biomolecular Building Blocks (Cont’d) DNAs and RNAs DNA structure Difference between DNA and RNA https://pmgbiology.com/tag/nucleotide/ https://theory.labster.com/structure-dna/ https://byjus.com/biology/difference-between-dna-and-rna/ Biomolecular Building Blocks (Cont’d) ChE 475 Biochemical Engineering II Carbohydrates and Fats Polysaccharides form by linking monosaccharides via glycosidic bonds, releasing H₂O Triacylglycerol (TAG) is formed by the esterification of three fatty acid molecules with one glycerol molecule https://www.shutterstock.com/image-vector/vector-set-three-categories-carbohydrates-monosaccharide-2251483619 Guerrero, Alicia. (2017). The dietary and thermoregulatory role of blubber as revealed by fatty acids. 10.13140/RG.2.2.16285.97765. © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 15, 2025 © 2025 by Oindrila Hossain, PhD Enzymes and Enzyme Kinetics ChE 475 Biochemical Engineering II Enzymes ▪ Enzymes- Highly specific biological catalysts - Special proteins that speed up chemical reactions by lowering the activation energy ▪ Enzymes helps in digestion, metabolism, DNA replication, and cell signaling. ▪ Enzymes are classified into six major types based on the type of reaction they catalyze- Acronym "O T H L I L“ Chloramphenicol Acetyltransferase (CAT) Enzyme HW- Find out the six types of enzymes and their functionality. https://www.verywellhealth.com/enzymes-8705866 Sarebanhassanabadi, Mohammadtaghi. (2014). Designing a Hydrogen Peroxide Biosensor Using Catalase and Modified Electrode with Magnesium Oxide Nanoparticles. International journal of electrochemical science. 257 - 271. © 2025 by Oindrila Hossain, PhD Enzymes and Enzyme Kinetics (Cont’d) ChE 475 Biochemical Engineering II Working Principle of Enzymes Amylase: A digestive enzyme that breaks down starches Creatine kinase (CK): Helps muscles create energy DNA polymerase: Facilitates growth by allowing DNA to duplicate https://www.verywellhealth.com/enzymes-8705866 © 2025 by Oindrila Hossain, PhD Enzymes and Enzyme Kinetics (Cont’d) ChE 475 Biochemical Engineering II Michaelis-and-Menten Kinetics Assumptions ▪ Steady-state: Formation and breakdown of the ES complex are balanced. ▪ Excess substrate: The substrate concentration is much higher than the enzyme concentration. ▪ Rapid equilibrium: The binding of the enzyme and substrate reaches equilibrium quickly . Michaelis-and-Menten equation: Significance of M-M Kinetics? https://tuitiontube.com/the-michaelis-menten-equation/ © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics Microbial Growth Phases Decline Phase Lag Phase: Bacteria adjust to their new environment; no significant cell division occurs Acceleration phase: Growth starts Log (Exponential) Phase: Rapid cell division; population doubles at a constant, exponential rate Decline phase: Growth rate slows Stationary Phase: Cell division rate equals the death rate, keeping population size stable Death Phase: Cell death exceeds growth; population declines until cells die or become dormant https://biologynotesonline.com/bacterial-growth-curve-protocol/ Acceleration Phase © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) A Real-world Microbiology Marvel- The Grand Prismatic Spring Thermophilic Bacteria thrives in high-temperature zones- often between 45°C and 80°C (sometimes even higher) Common genera: Thermus, Chloroflexus, Aquifex, Sulfolobus (archaea), etc. Spring Zone Temperature Range Likely Growth Phase Microbes Present Hot center (70– 90°C) Very high Exponential (if adapted), Lag (if not) Aquifex, Thermocrinis Orange/yellow edges (45–65°C) Moderatehigh Mostly Exponential or Stationary Chloroflexus, Thermus Cool outer edges Lower temps (30–45°C) Possibly mixed phases Cyanobacteria, others Images Captured by Dr. Noor Mohammad https://icjs.us/thermophilic-life-in-yellowstone-national-park-challenges-the-physical-and-chemical-limits-of-survival/ © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Key Ions That Modulate Growth Phases Ion Role Effect on Growth Phases Mg²⁺ Enzyme cofactor, ribosome & membrane stabilizer ? Ca²⁺ Cell wall stability, signaling, sporulation ↓ Lag (in some species), promotes sporulation in stationary phase K⁺ Osmoregulation, pH balance, enzyme activation Essential for exponential growth; deficiency prolongs lag Na⁺ Osmotic pressure, transport processes Needed in low levels; high Na⁺ can induce stress and prolong lag Redox reactions, cytochrome and enzyme cofactor Critical for respiration; deficiency ↓ exponential growth Mn²⁺ Cofactor in oxidative stress enzymes Affects survival in stationary phase; protects against ROS Zn²⁺ Cofactor for DNA-binding proteins, transcription factors Required for proper cell division, affects acceleration phase Cu²⁺ Redox cofactor, but toxic at high levels High Cu²⁺ can induce oxidative stress → early entry to stationary/death phase Co²⁺ Vitamin B₁₂ biosynthesis Affects anaerobic microbes, especially in industrial fermentations Ni²⁺ Urease cofactor, involved in some hydrogenases Important for certain bacteria (e.g., Helicobacter) Fe²⁺ / Fe³⁺ PO₄³⁻ ATP, nucleotides, phospholipids (Phosphate) Limiting PO₄³⁻ can trigger early stationary phase or secondary metabolite production © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Increase in Lag (h) Importance of Magnesium Ion Concentration of MgSO4 (mg/l) Why? © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Availability of Viable Cells Lag Phase: 𝑥 = 𝑥0 𝑥 = number of viable cells Log (Exponential) Phase: 𝑥 = 𝑥0 𝑒 𝜇𝑡 𝜇 = Specific growth rate Stationary Phase: 𝑥 = 𝑥0 + 𝑆0 𝑌𝑥/𝑠 𝑌𝑥/𝑠 = Yield Death Phase: 𝑥 = 𝑥0 𝑒 (𝜇−𝑘𝑑 )𝑡 𝑘𝑑 = Specific death rate constant © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 19, 2025 © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Availability of Viable Cells Lag Phase: 𝑥 = 𝑥0 𝑥 = number of viable cells Log (Exponential) Phase: 𝑥 = 𝑥0 𝑒 𝜇𝑡 𝜇 = Specific growth rate Stationary Phase: 𝑥 = 𝑥0 + 𝑆0 𝑌𝑥/𝑠 𝑌𝑥/𝑠 = Yield Death Phase: 𝑥 = 𝑥0 𝑒 (𝜇−𝑘𝑑 )𝑡 𝑘𝑑 = Specific death rate constant © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Availability of Viable Cells Log (Exponential) Phase: 𝑙𝑛2 Doubling time, 𝑡𝑑 = 𝜇 𝑥 = 𝑥0 𝑒 𝜇𝑡 𝜇 = Specific growth rate How? 𝜇𝑚𝑎𝑥 = max. specific growth rate 1 𝑑𝑥 = 𝑥 𝑑𝑡 How can we get 𝜇 from cell growth data? OD₆₀₀ (or biomass x) 0 0.10 2 0.22 4 0.48 6 0.95 ... ... Slope = 𝜇 lnx Time (h) lnx0 Time (h) © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Historical Background of Monod Growth Kinetics ▪ Pre-Monod Era (Before 1940s): - Microbial growth was known to depend on nutrients and environmental conditions, but early models were mostly empirical. - While growth phases were identified, specific growth rate wasn't yet mathematically related to substrate concentration. ▪ 1942 – Jacques Monod’s Breakthrough: - French biochemist Jacques Monod (Nobel Prize in Physiology or Medicine, 1965) published his seminal paper: “The Growth of Bacterial Cultures” (1942) - He proposed a mathematical relationship between the specific growth rate (µ) of microbes and substrate concentration (S), now famously known as the Monod Equation: 𝜇= 𝜇𝑚𝑎𝑥 𝑆 𝐾𝑠 + 𝑆 - The model was analogous to the Michaelis-Menten equation for enzyme kinetics © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Historical Background of Monod Growth Kinetics ▪ 1950s–60s – Adoption in Bioprocess Engineering ▪ 1970s–1980s – Environmental Applications ▪ 1990s–Present – Advanced Bioprocess Modeling, Synthetic Biology © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Monod Growth Kinetics Specific growth rate (μ) depends on the nutrient concentration (S) 𝜇= 𝜇𝑚𝑎𝑥 𝑆 𝐾𝑠 + 𝑆 𝐾𝑠 = half saturation constant Where, 𝜇 = 0.5𝜇𝑚𝑎𝑥 When S>>> 𝐾𝑠 what happens? ▪ When S is very high, cells are not limited by the substrate anymore. ▪ When the substrate concentration is at least 10 times higher than 𝐾𝑠 then μ is effectively equal to 𝜇𝑚𝑎𝑥 ▪ Adding more substrate won’t increase growth rate beyond this point. https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002/section002/black/page001.html © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Monod Growth Kinetics Specific growth rate (μ) depends on the nutrient concentration (S) 𝜇= 𝜇𝑚𝑎𝑥 𝑆 𝐾𝑠 + 𝑆 𝐾𝑠 = half saturation constant Where, 𝜇 = 0.5𝜇𝑚𝑎𝑥 When S<<< 10𝐾𝑠 what happens? ▪ The shift from exponential to stationary phase happens suddenly, without a gradual decline in growth rate. https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002/section002/black/page001.html © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Behavior in Different Substrate Range © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II A microorganism is grown in a 60 L batch bioreactor containing an initial glucose concentration of 200 g/L and an initial biomass concentration of 0.6 g/L. The culture exhibits a doubling time of 2.2 hours under optimal conditions. At t = 7 hours, it was found that: - Biomass = 4.2 g/L - Substrate concentration = 160 g/L - A metabolic by-product (lactic acid) has accumulated to 18 g/L, and growth begins to slow beyond this concentration. Questions: (a) Calculate the maximum specific growth rate, 𝜇𝑚𝑎𝑥 , assuming no inhibition up to 7 hours. (b) Determine the biomass yield coefficient, 𝑌𝑥/𝑠 , over the 7-hour period. (c) Estimate the total biomass (g) present in the reactor at 7 hours. (d) Assuming growth completely stops when lactic acid reaches 25 g/L, and that it accumulates linearly at the same rate as before, calculate when stationary phase will begin. (e) Compare this to a system without by-product inhibition. How much more biomass could theoretically be produced in that case? © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Other Kinetic Models Read about which one is used when Tsipa, Argyro & Varnava, Constantina & Grenni, Paola & Ferrara, V. & Pietrelli, Andrea. (2021). Bio-Electrochemical System Depollution Capabilities and Monitoring Applications: Models, Applicability, Advanced Bio-Based Concept for Predicting Pollutant Degradation and Microbial Growth Kinetics via Gene Regulation Modelling. Processes. 9. 1038. 10.3390/pr9061038. © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 21, 2025 © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Continuous Process 𝑥0 = 0, 𝑆0 , F A continuous fermenter (CSTR) is a bioreactor where fresh medium is continuously added, and culture liquid is continuously removed to keep the culture volume constant. Accumulation=Input+Generation−Output 𝑥, 𝑆, F At steady state, net accumulation Amount of cell at starting point (𝑥0 ) is negligible https://www.sciencedirect.com/topics/engineering/monod-expression © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Microbial Growth Kinetics (Cont’d) Continuous Process 𝑥0 = 0, 𝑆0 , F At steady state, 𝑥, 𝑆, F Derive these equations in 5 min https://www.sciencedirect.com/topics/engineering/monod-expression © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II Washout Condition At washout, biomass → 0, and substrate → 𝑆0 (incoming feed concentration) From the Monod equation: At washout, 𝜇 = 𝐷max , and 𝑆 = 𝑆0 (because no biomass is consuming it), so If substrate concentration is very high (i.e., S0 >>> 𝐾𝑠 ), then But that’s idealized. In practice, 𝐷max is slightly less than 𝜇max To avoid washout, D < Dmax < μmax https://www.sciencedirect.com/topics/engineering/monod-expression Why Can’t Cells Reproduce Fast Enough If 𝐷>μmax ? © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II How Monod Kinetics Connects to Aeration Tanks in ETPs Substrate (S) Organic pollutants in the water that microbes “eat” Microbial Growth (μ) Microbes multiply based on how much "food" is available Monod Equation More pollutant = faster growth, but only up to a limit (μmax) Low pollutant Microbes get hungry and slow down High pollutant Microbes are happy and grow near their max speed https://www.shutterstock.com/image-vector/wastewater-aeration-systems-info-isometric-cartoon-2203214447 Aeration tank in Renata Pharma, Kashor Site © 2025 by Oindrila Hossain, PhD Microbial Growth Kinetics (Cont’d) ChE 475 Biochemical Engineering II In an aeration tank designed for biological wastewater treatment, the initial pollutant concentration in the influent is 500 mg/L. The system operates at a dilution rate of 0.2 h⁻¹, with a maximum specific growth rate of 0.8 h⁻¹ and a Monod constant of 50 mg/L. The yield coefficient is 0.5 g biomass/g pollutant removed. According to the Department of Environment (DoE) guidelines, the permissible pollutant level in the treated effluent is <30 mg/L. Questions: (a) Determine whether the current operating conditions can meet the DoE requirement for BOD in the effluent. (b) If yes, calculate how much biomass concentration (in mg/L) is needed in the aeration tank to meet this treated BOD level. © 2025 by Oindrila Hossain, PhD Ways of Measuring Biomass ChE 475 Biochemical Engineering II 1. Direct Methods ▪ Dry Weight Measurement: - Dry cells at ~105°C until constant weight. - Accurate but time-consuming. ▪ Ash-Free Dry Weight (AFDW): - Combust dried biomass at 550°C. - Removes inorganic content. 2. Indirect Methods ▪ Optical Density (OD600): - Quick estimate of biomass. - Needs calibration. © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Ways of Measuring Biomass (Cont’d) 2. Indirect Methods ▪ Microscopic Counting (Hemocytometer): - Count total cells using a grid. - Live/dead distinction requires staining. ▪ Colony Forming Units (CFU): - Viable cells form colonies. - Requires incubation (24–48 h). 3. Biochemical-Based Methods- Read Hemocytometer Colony Counting https://www.labtestsguide.com/hemocytometer © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 22, 2025 © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Bioreactor vs Fermenter Feature Fermenter Bioreactor Definition Vessel for microbial fermentation. System supporting any biological reaction. Organisms Used Only fermentative microbes (bacteria, yeast, fungi). Unicellular microbes, plant, animal cells, enzymes. Products Alcohol, antibiotics, acids. Vaccines, insulin, monoclonal antibodies, enzymes. Design Consideration Rugged – can handle shear and foam. Gentle – low shear, high oxygen transfer efficiency. Oxygen Requirements Only anaerobic Can operate both anaerobic and aerobic conditions Process Complexity Moderate. High – requires tight environmental control. Examples Ethanol from Saccharomyces cerevisiae. mAbs from CHO cells, insulin from E. coli. https://www.differencebetween.com/difference-between-bioreactor-and-vs-fermentor/ © 2025 by Oindrila Hossain, PhD Importance of Fermentation Reaction ChE 475 Biochemical Engineering II ▪ Cell respiration is a controlled breakdown of sugars using redox reactions. ▪ It efficiently captures energy to make ATP, unlike combustion which just produces heat. https://old-ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled/energy-conversions.html © 2025 by Oindrila Hossain, PhD Products from Fermentation Reaction https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_%28Hartline%29/01%3A_Labs/1.22%3A_Fermentation ChE 475 Biochemical Engineering II © 2025 by Oindrila Hossain, PhD Applications of Bioreactor ChE 475 Biochemical Engineering II ▪ Ethanol production by fermentation ▪ Production of other biofuels ▪ Production of antibodies ▪ Production of citric acid ▪ Production of lactic acid ▪ Production of other Important chemicals Jain, Sapna & Pant, Kamal & Kassaye, Samuel & Singh, Shree. (2017). Nanocatalysts: Green Fuel and Chemical Production. 10.1081/E-ENN3-120053518. © 2025 by Oindrila Hossain, PhD Biorefinery ChE 475 Biochemical Engineering II ▪ A biorefinery is an integrated facility that converts biomass into fuels, energy, and value-added chemicals and materials. ▪ Think of it as the bio-based equivalent of a petroleum refinery. ▪ Feedstocks Used- Lignocellulosic biomass – crop residues, wood chips, grasses - Sugary/starchy crops – sugarcane, corn, cassava - Oily feedstocks – soybean, palm, algae. - Organic waste – food waste, municipal waste, manure. Type Example Whole Crop Biorefinery Corn biorefinery → ethanol, corn oil, DDGS Lignocellulosic Biorefinery Straw → ethanol, lignin → biochar Green Biorefinery Fresh grass → protein concentrate, lactic acid Marine Biorefinery Algae → biodiesel, omega-3, pigments https://www.biorender.com/template/biorefinery-46106 ▪ Core Processes- Mechanical – grinding, drying Chemical – hydrolysis, trans-esterification -Biological – fermentation, enzymatic digestion - Thermochemical – pyrolysis, gasification. © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) https://documents.parliament.qld.gov.au/com/PWUC-0F0F/RN855PLFSE-599B/que-19Oct2015-bio.pdf ChE 475 Biochemical Engineering II © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Biorefinery (Cont’d) Gasification ▪ Definition: High-temperature partial oxidation of biomass ▪ Temp. Range: 700–1000°C ▪ Atmosphere: Limited O2 (sub-stoichiometric) ▪ Main Product: Syngas (CO + H2) Dioxin ▪ Reaction: CₓHᵧ + O₂ → CO + H₂ + others ▪ Applications: Electricity, Hydrogen, Synthetic fuels (Fischer-Tropsch) ▪ Advantages: Cleaner process than incineration; no dioxin formation due to oxygen-deficient environment and particulate removal ▪ Flexibility: Syngas can be processed before combustion https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Liquefaction (HTL) ▪ Definition: Biomass to bio-oil in hot, pressurized water ▪ Conditions: 250–350°C, 10–25 MPa ▪ No drying needed → good for wet algae ▪ Products: Bio-crude ▪ Applications: Green diesel, Jet fuel, Industrial lubricants ▪ Advantages: Can process wet biomass, reducing the need for drying, produces bio-crude that can be refined into fuels ▪ Challenges: High energy demand, equipment corrosion due to water pressure, and cost of maintaining high-pressure conditions. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Pyrolysis ▪ Definition: Thermal decomposition without oxygen ▪ Temp Range: 350–600°C ▪ Products: Bio-oil (liquid), Charcoal (solid), Syngas (minor) ▪ Types: Fast pyrolysis → bio-oil; Slow pyrolysis → charcoal ▪ Applications: Bio-oil for fuel, Char as soil enhancer (biochar) ▪ Advantages: Enables use of larger biomass particles; lower pre-treatment costs ▪ Challenges: Energy-intensive, produces acidic bio-oil, which requires costly storage and handling ▪ By-product: Bio-char, usable as solid fuel or fertilizer. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Direct Combustion ▪ Definition: Complete combustion of biomass in the presence of oxygen to produce heat and ash ▪ Temp Range: 800–1000°C ▪ Products: Heat energy (primary), Flue gases (CO₂, H₂O), Ash (solid residue) ▪ Purpose: Mainly used for energy recovery (electricity, heat) ▪ Applications: Heat for industrial processes, steam generation, electricity in CHP systems ▪ Advantages: Volume reduction of biomass waste (up to 90%), Immediate energy generation, Can handle mixed or contaminated biomass ▪ Challenges: Produces air pollutants (dioxins, NOₓ, particulates), Not suitable for wet biomass, No bio-oil or char products ▪ By-product: Ash, which can be used in construction or as a soil amendment. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Anaerobic Digestion of Biomass to Produce Biogas ▪ Anaerobic digestion (AD) is a biological process that breaks down organic material in the absence of oxygen to produce biogas, mainly methane (CH₄) and carbon dioxide (CO₂). ▪ Four Stages: - Hydrolysis: Complex organics → simpler compounds - Acidogenesis: Simpler compounds → VFAs, H₂, CO₂ - Acetogenesis: VFAs → acetate, H₂, CO₂ - Methanogenesis: Acetate, H₂ → CH₄, CO₂ ▪ Conditions: 30–60°C, pH: 6.5–8, Retention: 20–40 days ▪ Biogas composition: 50–70% CH₄, 30–50% CO₂ + trace gases ▪ Purpose: Biogas for energy, digestate for fertilizer ▪ Bacteria usually come with biomass like manure or food waste Summarized from https://en.wikipedia.org/wiki/Anaerobic_digestion Terziev, A.; Zlateva, P.; Ivanov, M. Enhancing the Fermentation Process in Biogas Production from Animal and Plant Waste Substrates in the Southeastern Region of Bulgaria. Fermentation 2024, 10, 187. https://doi.org/10.3390/fermentation10040187 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Bioethanol Production Commercial bioethanol can be produced from various types of feedstock, including sugarcane, sugar beets, corn (maize), wheat, and potatoes https://www.youtube.com/watch?v=dG2_-OhnNGs © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production Biodiesel is produced from vegetable oils, animal fats, or used cooking oils through a process called transesterification. The production involves: ▪ Pre-treatment: Removing water, solids, and free fatty acids to prepare clean oil. ▪ Transesterification: Reacting the oil with methanol and a catalyst (e.g. NaOH) to convert triglycerides into biodiesel (methyl esters) and glycerol. ▪ Separation: Allowing the biodiesel and glycerol to separate by settling. ▪ Purification: Washing and drying the biodiesel to remove any impurities. https://farmdocdaily.illinois.edu/2023/02/biodiesel-and-renewable-diesel-whats-the-difference.html © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II ChE 475 Biochemical Engineering II Oindrila Hossain, PhD Lecturer Department of Chemical Engineering Bangladesh University of Engineering and Technology April 26, 2025 © 2025 by Oindrila Hossain, PhD RECAP Bioreactor vs Fermenter ChE 475 Biochemical Engineering II Feature Fermenter Bioreactor Definition Vessel for microbial fermentation. System supporting any biological reaction. Organisms Used Only fermentative microbes (bacteria, yeast, fungi). Unicellular microbes, plant, animal cells, enzymes. Products Alcohol, antibiotics, acids. Vaccines, insulin, monoclonal antibodies, enzymes. Design Consideration Rugged – can handle shear and foam. Gentle – low shear, high oxygen transfer efficiency. Oxygen Requirements Only anaerobic Can operate both anaerobic and aerobic conditions Process Complexity Moderate. High – requires tight environmental control. Examples Ethanol from Saccharomyces cerevisiae. mAbs from CHO cells, insulin from E. coli. https://www.differencebetween.com/difference-between-bioreactor-and-vs-fermentor/ © 2025 by Oindrila Hossain, PhD Importance of Fermentation Reaction RECAP ChE 475 Biochemical Engineering II ▪ Cell respiration is a controlled breakdown of sugars using redox reactions. ▪ It efficiently captures energy to make ATP, unlike combustion which just produces heat. https://old-ib.bioninja.com.au/higher-level/topic-8-metabolism-cell/untitled/energy-conversions.html © 2025 by Oindrila Hossain, PhD Products from Fermentation Reaction https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_%28Hartline%29/01%3A_Labs/1.22%3A_Fermentation RECAP ChE 475 Biochemical Engineering II © 2025 by Oindrila Hossain, PhD Applications of Bioreactor RECAP ChE 475 Biochemical Engineering II ▪ Ethanol production by fermentation ▪ Production of other biofuels ▪ Production of antibodies ▪ Production of citric acid ▪ Production of lactic acid ▪ Production of other Important chemicals Jain, Sapna & Pant, Kamal & Kassaye, Samuel & Singh, Shree. (2017). Nanocatalysts: Green Fuel and Chemical Production. 10.1081/E-ENN3-120053518. © 2025 by Oindrila Hossain, PhD Biorefinery RECAP ChE 475 Biochemical Engineering II ▪ A biorefinery is an integrated facility that converts biomass into fuels, energy, and value-added chemicals and materials. ▪ Think of it as the bio-based equivalent of a petroleum refinery. ▪ Feedstocks Used- Lignocellulosic biomass – crop residues, wood chips, grasses - Sugary/starchy crops – sugarcane, corn, cassava - Oily feedstocks – soybean, palm, algae. - Organic waste – food waste, municipal waste, manure. Type Example Whole Crop Biorefinery Corn biorefinery → ethanol, corn oil, DDGS Lignocellulosic Biorefinery Straw → ethanol, lignin → biochar Green Biorefinery Fresh grass → protein concentrate, lactic acid Marine Biorefinery Algae → biodiesel, omega-3, pigments https://www.biorender.com/template/biorefinery-46106 ▪ Core Processes- Mechanical – grinding, drying Chemical – hydrolysis, trans-esterification -Biological – fermentation, enzymatic digestion - Thermochemical – pyrolysis, gasification. © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) https://documents.parliament.qld.gov.au/com/PWUC-0F0F/RN855PLFSE-599B/que-19Oct2015-bio.pdf RECAP ChE 475 Biochemical Engineering II © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II RECAP Gasification ▪ Definition: High-temperature partial oxidation of biomass ▪ Temp. Range: 700–1000°C ▪ Atmosphere: Limited O2 (sub-stoichiometric) ▪ Main Product: Syngas (CO + H2) Dioxin ▪ Reaction: CₓHᵧ + O₂ → CO + H₂ + others ▪ Applications: Electricity, Hydrogen, Synthetic fuels (Fischer-Tropsch) ▪ Advantages: Cleaner process than incineration; no dioxin formation due to oxygen-deficient environment and particulate removal ▪ Flexibility: Syngas can be processed before combustion https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Liquefaction (HTL) ▪ Definition: Biomass to bio-oil in hot, pressurized water ▪ Conditions: 250–350°C, 10–25 MPa ▪ No drying needed → good for wet algae ▪ Products: Bio-crude ▪ Applications: Green diesel, Jet fuel, Industrial lubricants ▪ Advantages: Can process wet biomass, reducing the need for drying, produces bio-crude that can be refined into fuels ▪ Challenges: High energy demand, equipment corrosion due to water pressure, and cost of maintaining high-pressure conditions. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Pyrolysis ▪ Definition: Thermal decomposition without oxygen ▪ Temp Range: 350–600°C ▪ Products: Bio-oil (liquid), Charcoal (solid), Syngas (minor) ▪ Types: Fast pyrolysis → bio-oil; Slow pyrolysis → charcoal ▪ Applications: Bio-oil for fuel, Char as soil enhancer (biochar) ▪ Advantages: Enables use of larger biomass particles; lower pre-treatment costs ▪ Challenges: Energy-intensive, produces acidic bio-oil, which requires costly storage and handling ▪ By-product: Bio-char, usable as solid fuel or fertilizer. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Direct Combustion ▪ Definition: Complete combustion of biomass in the presence of oxygen to produce heat and ash ▪ Temp Range: 800–1000°C ▪ Products: Heat energy (primary), Flue gases (CO₂, H₂O), Ash (solid residue) ▪ Purpose: Mainly used for energy recovery (electricity, heat) ▪ Applications: Heat for industrial processes, steam generation, electricity in CHP systems ▪ Advantages: Volume reduction of biomass waste (up to 90%), Immediate energy generation, Can handle mixed or contaminated biomass ▪ Challenges: Produces air pollutants (dioxins, NOₓ, particulates), Not suitable for wet biomass, No bio-oil or char products ▪ By-product: Ash, which can be used in construction or as a soil amendment. https://www.intechopen.com/chapters/78641 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Anaerobic Digestion of Biomass to Produce Biogas ▪ Anaerobic digestion (AD) is a biological process that breaks down organic material in the absence of oxygen to produce biogas, mainly methane (CH₄) and carbon dioxide (CO₂). ▪ Four Stages: - Hydrolysis: Complex organics → simpler compounds - Acidogenesis: Simpler compounds → VFAs, H₂, CO₂ - Acetogenesis: VFAs → acetate, H₂, CO₂ - Methanogenesis: Acetate, H₂ → CH₄, CO₂ ▪ Conditions: 30–60°C, pH: 6.5–8, Retention: 20–40 days ▪ Biogas composition: 50–70% CH₄, 30–50% CO₂ + trace gases ▪ Purpose: Biogas for energy, digestate for fertilizer ▪ Bacteria usually come with biomass like manure or food waste Summarized from https://en.wikipedia.org/wiki/Anaerobic_digestion Terziev, A.; Zlateva, P.; Ivanov, M. Enhancing the Fermentation Process in Biogas Production from Animal and Plant Waste Substrates in the Southeastern Region of Bulgaria. Fermentation 2024, 10, 187. https://doi.org/10.3390/fermentation10040187 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Bioethanol Production Commercial bioethanol can be produced from various types of feedstock, including sugarcane, sugar beets, corn (maize), wheat, and potatoes https://www.youtube.com/watch?v=dG2_-OhnNGs © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) RECAP ChE 475 Biochemical Engineering II Biodiesel Production Biodiesel is produced from vegetable oils, animal fats, or used cooking oils through a process called transesterification. The production involves: ▪ Pre-treatment: Removing water, solids, and free fatty acids to prepare clean oil. ▪ Transesterification: Reacting the oil with methanol and a catalyst (e.g. NaOH) to convert triglycerides into biodiesel (methyl esters) and glycerol. ▪ Separation: Allowing the biodiesel and glycerol to separate by settling. ▪ Purification: Washing and drying the biodiesel to remove any impurities. https://farmdocdaily.illinois.edu/2023/02/biodiesel-and-renewable-diesel-whats-the-difference.html © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production https://letstalkscience.ca/educational-resources/stem-explained/algae-biofuel-can-pond-scum-power-planet © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production from Algae ▪ Algae produce several times more oil than palm feedstock. ▪ Up to 90% less water, no need for arable land ▪ 33–50% lipids for biodiesel; sugars/proteins for bioethanol ▪ Ideal for biodiesel and jet fuel where few alternatives exist ▪ Can integrate with waste treatment or CO₂-rich industrial systems ▪ Using saline/wastewater enhances sustainability ▪ Co-product use (proteins, pigments) boosts economic and environmental viability https://www.freepik.com/premium-photo/algae-fuel-biofuel-industry-lab-researching-alternative-fossil-algae-fuel-algal-biofuel_29157350.htm © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production from Algae Bošnjaković, M.; Sinaga, N. The Perspective of Large-Scale Production of Algae Biodiesel. Appl. Sci. 2020, 10, 8181. https://doi.org/10.3390/app10228181 © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production from Algae Can algae-based biofuel have higher carbon footprint than conventional fuel? https://www.globalseafood.org/advocate/farming-algal-fuel-economics-challenge-process-potential/ar https://e360.yale.edu/digest/algae-biofuel-carbon-footprint © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Biodiesel Production from Algae Open pond vs. Closed system ▪ Cost ▪ Productivity ▪ Contamination ▪ Environmental Control ▪ Water & CO₂ Use ▪ Sustainability ▪ Application Which one is better? © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Antibodies Production https://www.yourgenome.org/theme/how-is-dna-turned-into-protein-the-central-dogma-of-molecular-biology/ © 2025 by Oindrila Hossain, PhD Biorefinery (Cont’d) ChE 475 Biochemical Engineering II Citric Acid Production https://www.sciencedirect.com/science/article/pii/S095965261934185X © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Biorefinery (Cont’d) Lactic Acid Production ? https://www.sciencedirect.com/science/article/abs/pii/S2352186420316370 © 2025 by Oindrila Hossain, PhD Requirements of Bioreactors ChE 475 Biochemical Engineering II ▪ Sterile environment to prevent contamination ▪ Ensure homogeneity of the culture medium ▪ Optimize agitation rate to minimize shear damage ▪ High oxygen transfer rate (OTR) for aerobic processes ▪ Ensure proper nutrients are available. ▪ Efficient removal of metabolic by-products ▪ pH control to ensure organisms thrive ▪ Gentle heat transfer https://www.sciencedirect.com/science/article/abs/pii/S2352186420316370 © 2025 by Oindrila Hossain, PhD Bioreactors Configurations ChE 475 Biochemical Engineering II Stirred Tank Reactor Most widely used bioreactor type, consisting of a tank with impellers for mechanical agitation, aeration, and uniform mixing. Ideal for aerobic fermentation processes like ethanol, citric acid, and antibiotic production Key Features: ▪ Impellers (various shapes/sizes) and baffles enhance mixing and prevent vortex formation ▪ Air sparging supplies oxygen for aerobic processes ▪ Aspect ratio (H/D) is usually chosen >1 Why ▪ Includes temperature and pH control, sampling ports, and internal cooling coils. Operating Volume: Filled to 70–80% of capacity Why https://www.sciencedirect.com/topics/engineering/stirred-vessel © 2025 by Oindrila Hossain, PhD Bioreactors Configurations (Cont’d) ChE 475 Biochemical Engineering II Stirred Tank Reactor Applications: Suitable for free and immobilized enzyme reactions, plant and animal cell cultures, and biopharmaceutical production Advantages: ▪ Excellent mixing and mass transfer ▪ Versatile for various fermentation types ▪ Easily scalable from lab to industry Limitations: ▪ Mechanical/shear stress may damage sensitive cells or catalysts ▪ High energy demand due to continuous agitation and aeration https://www.sciencedirect.com/topics/engineering/stirred-vessel © 2025 by Oindrila Hossain, PhD Bioreactors Configurations (Cont’d) ChE 475 Biochemical Engineering II Bubble Column Reactor A Bubble Column Reactor (BCR) is a cylindrical vessel where gas sparging provides aeration and mixing. It requires less energy than mechanical stirring and operates without moving parts Key Features: ▪ Gas introduced via a sparger at the bottom ▪ No moving parts ▪ Horizontal plates in tall columns to improve gas-liquid contact ▪ Height/diameter ratio typically >2 Operating Conditions: ▪ Low gas flow: Homogeneous flow with uniform bubble rise ▪ High gas flow: Heterogeneous flow with chaotic circulation ▪ Foam management: Requires antifoam or dispersers Collected from Web © 2025 by Oindrila Hossain, PhD Bioreactors Configurations (Cont’d) ChE 475 Biochemical Engineering II Bubble Column Reactor Applications: Yeast, beer, vinegar production, wastewater treatment, biogas production. Advantages: ▪ Low energy use ▪ Low capital cost ▪ Simple maintenance Limitations: ▪ Foam formation ▪ Flow issues at high gas velocities ▪ Gas phase backmixing Collected from Web © 2025 by Oindrila Hossain, PhD ChE 475 Biochemical Engineering II Bioreactors Configurations (Cont’d) Bubble Column Reactor ▪ Low gas flow rates: Small, uniform bubbles create homogeneous flow ▪ Increasing gas flow: Leads to turbulent flow with larger, chaotic bubbles ▪ Higher flow rates: Results in churn-turbulent flow, characterized by irregular circulation and bubble coalescence ▪ Flow behavior impacts: Affects mass transfer and reactor performance. https://translate.academic.ru/mist%20flow/en/xx/ © 2025 by Oindrila Hossain, PhD Bioreactors Configurations (Cont’d) ChE 475 Biochemical Engineering II Bubble Column Reactor Heterogeneous Flow in Bubble Column Reactor: Upward liquid velocity (𝑢𝐿) is influenced by both the column diameter (𝐷) and superficial gas velocity (𝑢G) Valid for: Mixing time tm can be obtained as: © 2025 by Oindrila Hossain, PhD Feature SHF SSF CBP Process Steps Separate hydrolysis and fermentation Simultaneous hydrolysis and fermentation Single-step hydrolysis and fermentation Time Long (separate steps) Shorter than SHF Shortest (integrated process) Cost High (requires two reactors) Lower (single reactor) Lowest (simplified process) Microbial Requirements Separate microbes for each step One microorganism for both functions Genetically engineered microorganism Control Easier control per step More complex due to dual functions Most complex due to dual optimization Yield Medium Higher than SHF Highest (integrated process) By-product Inhibition Potential inhibition from intermediates Less inhibition, but still possible Minimal inhibition due to integration Microbe/Enzyme Compatibility Easier optimization Harder due to temperature differences Requires specialized engineered strains
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