Bioprocess Engineering Flashcards
Stages of bioprocessing
initial purification
- bioreactor
- cell disruption
- first purification step to obtain clarified feed
- solvent exchange
intermediate purification
- chromatographic separation
final purification
- other separation operations
Cell Growth
- Cells selectively take up dissolved nutrients from the medium for energy production, biosynthesis and product formation.
- Growth is a result of both replication and change in cell size.
ΣS + X → ΣP + nX - Substrates + Cells → Extracellular products + More Cells
- Autocatalytic reaction → a reaction requiring no further changes of environment to continue to completion.
microbial Growth
Microbial growth
The rate of microbial growth is characterised by the net
specific growth rate, µnet,
1. µnet = (1/X)(dX/dt)
µnet = µg − kd
where X = cell mass concentration (g/l),
t = time (h),
µ net = net specific growth rate (h-1),
µ g = gross specific growth rate (h-1),
kd = rate of loss of cell mass due to cell death or endogenous metabolism* (h-1).
*Endogenous metabolism: cell catabolises cellular reserves for new building blocks and for energy-producing monomers.
Batch Culture
Culturing cells in a vessel with an initial charge of medium that is not altered by further nutrient addition or removal.
- lag phase
- exponential phase
- deceleration
- stationary
- death/decline
Lag Phase
- Period of adaptation of cells to a new environment.
- Cell mass may increase slightly, but no increase in cell number.
- Duration can be minimised by:
(a) adapting cells to growth medium/conditions pre-inoculation,
(b) use of young/active cells,
(c) larger inoculum size (5-10% by volume),
(d) optimising nutrient medium.
Exponential Phase
- Cell mass and number increase exponentially with time,
- All cell components grow at the same rate; (net specific growth rate, µnet = constant).
- Growth rate is independent of nutrient concentration.
- Average cell composition and size will not change with time.
- Cellular metabolic control system achieve maximum rates of reproduction.
X = X0(e^unet*t), where X is cell concentration at time t.
doubling time = ln2/unet or 0.693/unet or ln2/replication rate
Deceleration Phase
- Growth decelerates due to:
(a) depletion of ≥1 essential nutrients,
(b) accumulation of toxic by-products of growth. - Unbalanced growth
(i.e. cell composition and size will change; τd ≠ τ’d). - Stresses induced by (a) or (b) cause cell restructuring to increase cellular survival in a hostile environment.
Stationary Phase
- Net growth rate, µnet = 0 (i.e. no cell division) or µg = kd
growth rate = death rate. - One or more of the following may take place:
(a) Total [cell mass] may stay constant, but the no. of viable cells may decrease.
(b) Cell lysis may occur and viable cells may drop; a second growth phase may occur and cells may grow on lysis
products of lysed cells (cryptic growth).
(c) Cells may not be growing but have active metabolism to produce secondary metabolites. - Endogenous Metabolism
- Cell catabolises cellular reserves for new building blocks and
energy-producing monomers. - Maintenance Energy
- Essential metabolic functions (motility and cell damage repair).
- Conversion of cell mass into maintenance energy (or loss of cell mass due to cell lysis during stationary phase).
Yield
Yield – product amount formed per amount of reactant provided.
Theoretical (stoichiometric, true) Yield
= Total product formed(mass or moles)/Reactant used to form the product(mass or moles)
= Apparent Yield (observed)
Product present(mass or moles)/Reactant consumed (mass or moles)
When reactants or products are involved in additional reactions, the apparent yield may be different from the theoretical yield.
Apparent yield factors
At the end of the batch growth, we have an apparent(observed) growth yield (not a true constant because culture conditions can alter patterns of substrate utilisation).
For e.g., with a substrate that is both a carbon and energy source (e.g. glucose), it may be consumed as:
∆S = ∆Sbiomass assimilation
+ ∆S product assimilation
+ ∆S growth energy
+ ∆S maintenance energy
Maintenence coefficient
m, = specific rate of substrate
uptake for cellular maintenance.
m = ([dS/dt]m)/X
- When little external substrate is available (e.g., stationary phase), endogenous metabolism of biomass components is used for maintenance energy.
- Cellular maintenance ≅ energy expenditure to
(i) repair damaged cellular components,
(ii) transfer some nutrients and products in and out of cells,
(iii) motility,
(iv) adjust osmolarity of cells’ interior volume.
Microbial Products
Growth-associated products are produced simultaneously with microbial growth.
- Specific rate of product formation ∝ specific rate of growth, µg
Non-growth-associated product formation takes place during the stationary phase, when growth rate = 0.
- Many secondary metabolites (e.g. antibiotics) are non-growth associated products.
Mixed-growth-associated product formation takes place during slow growth and stationary phases.
Temperature
- As T increases toward optimal growth temperature, growth
rate ~ doubles for every 10°C increase. - > Topt range, growth rate decreases, thermal death may
occur.
− At high T, thermal death rate exceeds growth rate, causing a
net decrease in [viable cell]. - affects YX/S, and product formation.
e.g., when T > Topt range, maintenance requirements of cells increase (i.e. maintenance coefficient, m, increases), thus decreasing YX/S.
pH
- affects activity of enzymes, thus the microbial growth rate.
- optimal pH for growth rate may be different from that for product formation.
- pH deviation from optimal value, increases maintenance energy requirements.
- can adapt to expand pH range
- pH fluctuations depend on:
(i) nature of nitrogen source,
(ii) production of organic acids,
(iii) utilisation of acids (e.g. amino acids),
(iv)production of bases,
(v) presence of CO2. - pH control by buffer or active pH control system is key.
Dissolved oxygen
- Important substrate in aerobic fermentation.
- At high [cell], rate of O2 consumption may exceed rate of O2 supply.
- When O2 is the growth rate-limiting factor (i.e. when DO level is below the critical [DO]), specific growth rate varies with [DO] according to saturation kinetics. Below critical [O2], growth shows dependence on [DO]. Above a critical [O2], growth rate is independent of [DO].
- Critical [O2] ~ 5-10% and 10-50% of saturated [DO] for bacteria and yeast, and mold cultures, respectively.
