Large-Scale Bioreactors & Cell-Cultivation Processes – Comprehensive Lecture Notes

Introduction

• Purpose of the lecture: provide a panoramic introduction to large-scale bioreactors used to cultivate living cells for commercial, environmental and therapeutic purposes.

• Emphasis on: diversity of reactor formats, cell types, operating modes, regulatory pressures, real-world industrial examples, and recent trends (e.g., single-use systems).

Major Application Areas & Products

• Brewing & winemaking

  • Largest global cell-culture operations; vessels >10^5 L.

• Waste-water treatment

  • Semi-continuous/continuous mixed-culture systems for nutrient removal and resource recovery.

• Industrial & domestic enzymes (bacterial, fungal, yeast, other hosts).

• Baker’s yeast (biomass as final product)

  • Multiple Australian plants; example: Rose Hill (Parramatta).

• Cultured meat

  • Example: Val Foods (Alexandria) authorised to sell quail; operates a 2\times 10^4\;\text{L} fermenter.

• Fuel-ethanol

  • ≥ 3 Australian plants; hundreds worldwide for biofuels & solvents.

• Micro-algal high-value products

  • Omega-3 oils, nutraceutical pigments (e.g.
    astaxanthin).

• Amino-acid production (bacterial processes).

• Vaccines & therapeutic proteins (viruses, VLPs, mAbs, cytokines, etc.).

Cell Types Routinely Cultivated at Scale

• Bacteria (e.g., E.\;coli, Bacillus spp.)

• Filamentous fungi (secondary metabolites, recombinant enzymes).

• Yeasts (biomass, ethanol, beverage alcohol, recombinant proteins).

• Algae (initially biofuels, now high-value compounds).

• Insect cells (baculovirus expression of proteins/VLPs).

• Mammalian cells

  • Historically for biopharma; now also food (cultured meat).

Core Design & Operating Requirements

• Must match biological characteristics:

  • Replication rate, shear sensitivity, morphology (planktonic vs.
    mycelial), cell-size, O₂ demand.

  • Surface attachment needs (e.g., mammalian micro-carrier cultures).

• Environmental control loops typically include:

  • Temperature, dissolved O₂, pH.

  • Feed-rate (for fed-batch), photon flux for phototrophs.

• Sterility

  • Prevention of ingress & co-cultivation by contaminants.

  • GMP (Good Manufacturing Practice) adds validation, documentation & change-control layers, mainly in therapeutic production.

• Re-use vs. single use

  • Traditional: stainless-steel, steam-in-place (SIP), clean-in-place (CIP).

  • Trend: disposable polymer bags to lower validation downtime & effluent burden.

• Materials & scale window

  • Plastic, glass, mild/stainless steel, concrete (waste systems).

  • Volume spectrum: mL-scale to >10^6\;\text{L}.

• Instrumentation depth rises with: value of product → food → therapeutics (highest).

Operating Modes of Bioreactors

Batch

• All nutrients loaded at t=0; inoculum pitched; no in/out flow during run.

• Unsteady-state: biomass ↑, substrate ↓, product ↑.

• Standard growth curve:

  1. Lag (adjustment)

  2. Log/exponential (\mu_{max})

  3. Stationary (nutrient limitation or inhibitory product e.g., ethanol)

  4. Decline, sporulation or diauxic shifts (secondary carbon source).

Fed-Batch (mentioned via Baker’s yeast)

• Controlled feed addition postpones limitation, increases titres.

Continuous Culture (Chemostat)

• After batch start-up, matched inlet/outlet flows: F{in}=F{out} keeps working volume constant.

• Achieves steady state where
  \mu = D = \frac{F}{V} (specific growth rate =\text{dilution rate}).

• Biological ceiling: replication kinetics limit max useful D.

• Extensions:

  • Two-stage series (different T, inducer, pH to decouple growth vs.
    production).

  • Cell recycle via centrifuge/hollow-fibre to intensify biomass (ethanol example).

Major Reactor Classes

Mechanically Stirred (Stirred-Tank Reactors, STR)

• Universally applicable; flexibility via impeller choice, baffles, gas spargers, cooling coils.

• Lab/pilot schematic highlights: agitator shaft, multi-impeller train, ring sparger, internal cooling loop, foam breaker, pH & O₂ probes, SIP ports.

Airlift/Tower Fermenters

• No moving parts; gas sparging establishes circulation.

• Lower shear → suitable for fragile or slower-growing cells.

• High aspect ratio (3–10 m tall or >60 m in extreme cases) → elevated hydrostatic P enhances O₂ solubility.

Cylindro-Conical Fermenters (Beer)

• Cylindrical body + 60° cone; cooling jackets.

• Lager yeast flocculates & settles in cone → easy yeast harvest (Vegemite feedstock) & beer removal.

Aerobic & Anaerobic Digesters

• Mixed-culture waste-water or food-waste treatment.

• Example flow-sheet: anaerobic → aerobic → settler, with activated-sludge recycle.

• Control often feed-rate-to-biogas-rate feedback (EarthPower site, Camellia).

Photobioreactors

• Constraint: photon delivery supersedes nutrient supply.

• Closed designs: glass/plastic tubes, flat panels, internally lit STRs; pros – sterility, CO₂ co-utilisation, water conservation.

• Open designs: raceway ponds (≈30 cm deep), coastal salt basins (WA Daneilla salina since 1970s).

• Trade-offs: lower CAPEX vs.
  contamination, evaporation & O₂ build-up.

Single-Use (Disposable) Systems

• Drivers: minimise SIP/CIP downtime, reduce validation & cross-batch contamination, cut WFI & chemical usage.

• Formats & scales:

  • Roller bottles (10s mL; early Amgen EPO).

  • Wave or rocking bags (≈200–500 L).

  • Stirred-bag bioreactors with magnetic or overhead drive (up to 2{,}000\;\text{L}; vendors: Merck Mobius, Sartorius Flexsafe).

• Environmental trade-off: polymer waste vs.
  water/caustic/steam savings.

Illustrative Industrial Case Studies

• Val Foods cultured-quail meat: 20\;000\;\text{L} mammalian STR in Alexandria, AU.

• Shoalhaven Starches/Bomaderry ethanol:

  • Wheat starch hydrolysis → fermenters → distillation.

  • >3\times10^8\;\text{L·a}^{-1} ethanol; CO₂ capture for beverage-grade gas; spent-grain to animal feed.

• EarthPower (Camellia):

  • Two 24\;000\;\text{m}^3 anaerobic digesters convert Sydney food waste → biogas, fertiliser, treated effluent.

  • Feed-rate governed by real-time biogas production.

• ICI 60-m Methanol-to-Single-Cell-Protein tower (Seventies-Eighties):

  • 2.5\times10^5\;\text{kg h}^{-1} medium, 1.4!\unicode{x2013}!2.0\times10^4\;\text{kg h}^{-1} methanol, 9.3\times10^4\;\text{m}^3\, \text{h}^{-1} air.

  • Structured like distillation column with perforated trays → airlift circulation.

  • Technically successful, ultimately uneconomic.

• Daniella salina open ponds (WA), plus raceways in Hawaii & California – pigment & nutraceutical production on non-arable coastal land.

Scale-Up, Materials & Utilities

• Volume tiers: lab mL → pilot 10–100 L → demo 1–20 m³ → commercial 20 m³ to >10^6\;\text{L}.

• Materials chosen by:

  • Corrosion, pressure, cleaning needs, thermal conductivity, cost.

  • Concrete acceptable for low-value waste treatment; stainless obligatory for GMP biologics.

• Utilities & control loops increase with titer/value:

  • Cooling water for metabolic heat (Q \propto \Delta H_{metabolism}), dissolved-O₂ cascade (RPM + gas-flow + O₂-enrichment), antifoam addition, level & pressure control, photon sensors (for algae).

Regulatory, Ethical & Sustainability Considerations

• Industrial & beverage: food-safety authorities, HACCP.

• Therapeutics: GMP, validation of cleaning, sterility assurance level (SAL), documentation, change-control.

• Environmental goals:

  • CO₂ capture (photobioreactors, ethanol CO₂ bottling).

  • Waste diversion from landfill (EarthPower).

  • Water & chemical usage trade-offs (single-use vs.
    SIP stainless).

• Ethical aspects of cultured meat: animal-free protein, consumer acceptance, licencing milestones (e.g., Australian approval).

Key Equations & Quantitative Relationships

• Dilution rate: D = \frac{F}{V} \;\; [\text{h}^{-1}]

• Steady-state chemostat: \mu = D

• Volumetric mass balance in continuous reactor: F{in} = F{out}

• Hydrostatic pressure benefit (airlift): P = \rho g h → higher h boosts c^*{O2}.

Summary & Take-Home Messages

• Large-scale cell culture is highly diversified—no single bioreactor suits all organisms or products.

• Design must integrate biology (shear, oxygen, photon needs), process economics, and regulatory expectations.

• Modes of operation (batch, fed-batch, continuous, perfusion) offer different control over growth, productivity and inhibition.

• Novel trends:

  • Single-use disposable reactors rising in biopharma to cut CIP/SIP costs and downtime.

  • Photobioreactors targeting carbon capture and specialty algal metabolites.

  • Intensification strategies (cell recycle, high-aspect airlifts, perfusion hollow-fibres) combat growth or inhibition bottlenecks.

• Real-world examples—from beer cones to 60 m SCP towers—illustrate the vast scale range and ingenuity of bioprocess engineering.