🌿 1. Biorefineries

Definition: A biorefinery is like a “green factory” — it sustainably converts biomass 🌾 (plants, waste, algae, etc.) into food, feed, chemicals, materials, and energy (fuels, power, heat). → Goal: Replace fossil resources with renewable carbon and close material loops.

Sustainable processing means:

• Non-toxic, recyclable, degradable products
• Minimal contaminants
• Avoid solvents and harsh chemicals

Generations of Biorefineries:

1. 1st Generation: Use food crops (e.g., corn → bioethanol). ⚠️ Competes with food supply.
2. 2nd Generation: Use non-food biomass (e.g., straw, wood, residues). ✅ Better sustainability, but harder to process (lignocellulose).
3. 3rd Generation: Use CO₂ as carbon source!
   • Powered by photoautotrophs (cyanobacteria, algae) 🌞
   • Or chemoautotrophs (hydrogen-oxidizing bacteria) ⚡
   • Combine electricity + microbes = electro-biorefineries

Examples:

• e-kerosene, e-ammonia, e-methane from CO₂ reduction and renewable energy
• “Power-to-X” projects (DTU): integrate renewable power with CO₂ conversion

Europe status:

~3777 biorefineries in the EU (as of Oct 2025). Largest hubs: France 🇫🇷, Germany 🇩🇪, Italy 🇮🇹, Spain 🇪🇸.

⚡ 2. Electromicrobiology

Definition: Study of microorganisms that exchange electrons with their environment via Extracellular Electron Transfer (EET) 🔋

Two key types:

• Exoelectrogenic → donate electrons by oxidizing organics (e.g., acetate, glucose)
• Electrotrophic → accept electrons to reduce CO₂ → simple organics or CH₄

How microbes “talk” electrically:

• Through conductive pili (nanowires) with cytochromes
• Via conductive minerals (magnetite) or activated carbon
• Even across centimeters — e.g., Cable bacteria discovered 2012 ⚡ → Link sulfide oxidation and oxygen reduction over long distances

Applications (METs – Microbial Electrochemical Technologies):

1. 🪫 MFCs (Microbial Fuel Cells) – microbes generate electricity
2. ⚗️ MECs (Microbial Electrolysis Cells) – apply current → make H₂ or CH₄
3. 🧪 MES (Microbial Electrosynthesis) – use CO₂ + electricity → chemicals → TRL ~4-5 (pilot stage)

Unwanted application: Biocorrosion 🧲 — biofilms accelerate local metal corrosion (pitting), unlike smooth oxygen corrosion.

💨 3. Anaerobic Digestion (AD)

Definition: Microbial process that decomposes organic matter without oxygen → produces biogas (CH₄ + CO₂) and digestate (fertilizer).

Versatile technology:

• Small rural digesters to huge industrial lagoons
• Inputs: manure, wastewater, food waste, algae, sludge
• Outputs: biomethane, fertilizer, electricity, biofuels

🔬 Steps of Anaerobic Digestion (“Microbial Food Web”)

1. Hydrolysis 🧬 Break polymers → sugars, amino acids, fatty acids
   • Rate-limiting step
   • Pretreatments (grinding, heat, acid) improve degradation
2. Acidogenesis (Fermentation) 🍋 Sugars → volatile fatty acids (VFAs), ethanol, lactate
   • Fastest step
   • Too much NH₄⁺ or VFA → inhibition
   • Common microbes: Clostridium, Streptococcus, Propionibacterium
3. Acetogenesis 🧪 VFAs → acetate + H₂ + CO₂
   • Needs syntrophy with hydrogenotrophic methanogens
   • Example: Syntrophobacter, Smithella
4. Methanogenesis 💨 Archaea convert acetate or H₂/CO₂ → CH₄
   • Acetoclastic (Methanothrix) and hydrogenotrophic (Methanolinea) types
   • Sensitive to pH (6.5–8) and inhibitors (NH₃, sulfide)

Biodegradability test: BMP (Biochemical Methane Potential) measures methane yield → checks substrate suitability.

⚙️ Operational Conditions

• Temperature: Psychrophilic (4–20°C), Mesophilic (20–40°C), Thermophilic (45–70°C) → ~35°C or 55°C typical
• SRT (Solids Retention Time): days solids stay in reactor
• HRT (Hydraulic Retention Time): water residence time
• OLR (Organic Loading Rate): kg VS/m³·day fed to reactor

🇩🇰 Danish Biogas Context

• ~680 WWTPs; ~57 have digesters
• Only ⅓ of sludge biogas potential used
• MiDAS surveys (Aalborg University):
  • Unique microbial communities per plant
  • Strongly influenced by temp, load, pre-treatment, pH
  • Stable but under-optimized performance

💭 Foaming Problem in AD

Foam = operational nightmare ☁️ → Blocks gas/sludge separation, lowers yield, costly cleaning

Causes: Filamentous microbes (mainly Candidatus Brevefilum fermentans)

• Found in foaming digesters
• Chloroflexi phylum, forms dense symbioses with methanogens
• Identified by FISH probes
• Marker species but no control method yet 😬

⚡ Exercise Example

Estimate how much sewage sludge is needed to produce electricity for one AI response. (Uses energy conversion and methane yield data.)

🧫 4. Biopolymers

Wastewater isn’t waste — it’s a resource! ♻️ Can recover biopolymers from microbial biomass in treatment plants.

Types:

• Polysaccharides: cellulose, starch, alginate, agar, chitosan
• Proteins: gelatin, collagen Uses: textiles, paper, food, cosmetics, construction, bioplastics, etc.

🧴 Why Bioplastics?

🌍 Plastic crisis: 25 Mt waste/year in EU+US

80 % landfilled or leaked to ocean Petroleum plastics are cheap (~1 €/kg) but not sustainable. Bioplastics are biodegradable but costly (2–6 €/kg).

🧫 Polyhydroxyalkanoates (PHA)

Microbial storage polymers = natural bioplastics! Produced by bacteria (pure or mixed cultures). First full-scale mixed-culture PHA plant (Netherlands, 2025).

Properties:

• Linear polyesters, biodegradable, water-resistant
• Degrade to CO₂ + H₂O aerobically or CH₄ anaerobically
• Biocompatible (safe for medical use)

Two main forms:

• P(3HB): Poly-3-hydroxybutyrate
• P(3HV): Poly-3-hydroxyvalerate

Applications: Medicine 🩹, packaging 📦, drug carriers 💊, coatings 🎨.

⚗️ PHA Production Strategies

• Pure cultures: controlled but expensive
• Mixed cultures: cheaper, flexible, selected by reactor conditions
• Key process: Aerobic Dynamic Feeding (ADF) → feast/famine cycles
  • Long starvation → high PHA accumulation
  • Influenced by C-source, N/P absence, temperature

🧬 Extracellular Polymers (EPS) from Sludge

Activated sludge = treasure chest of biopolymers 💎 EPS = proteins, polysaccharides, glycoproteins, lipids, DNA

Kaumera biopolymer (Netherlands):

• Extracted from granular sludge
• Fire-resistant, biodegradable, smoke-free foam (🔥 withstands 1970°C!)
• Applications: composites, hydrogels, flame retardants

Global potential: 30 kg activated sludge per person/year → 50–100 Mt biopolymer/year 🌎

🔬 Toolbox for EPS Study

• Biochemistry: proteomics, glycomics, rheology
• Microbiology: metagenomics, genome-centric transcriptomics
• Chemistry: supramolecular and natural product analyses

Activated sludge still hides many unknown microbes and polymers — a frontier for biotechnology research.

✅ Summary Table