Cells and Cell Cultures

Transcription

1 Cells and Cell Cultures Beyond pure enzymes, whole cells are used and grown in biotechnological applications for a variety of reasons: cells may perform a desired transformation of a substrate, the cells themselves may be the desired product, or the cells may produce a desired product, such as penicillin. In this case, the desired product may be excreted, as for penicillin, and recovered in relatively simple fashion, or it can be retained within the cell, which then need to be lysed (ruptured) to recover the product from a complex mixture of cellular proteins. This approach is often needed for therapeutic proteins that are created by recombinant DNA technology. The resulting separation problem is one of the more challenging aspects of biochemical engineering. Generally, culture of the cells can be quite difficult experimentally and is even more demanding theoretically. L31-1

2 More about Metabolisms L31-2 All organisms have the same basic set of metabolic pathways, which consists of many coupled, interconnected reactions On a basic level, the metabolism consists of the catabolism (degradative pathways) and the anabolism (biosynthetic pathways) The oxidation of carbon fuels is an important source of cellular energy which drives the metabolism Organisms show a marked similarity in their major metabolic pathways Different organisms have different ways to obtain carbon and energy: carbon requirement: Autotrophs use Heterotrophs use energy source: Phototrophs use Chemotrophs use All animals belong to Plants belong to

3 L31-3 Metabolism Map (from: Kyoto Encyclopedia of Genes and Genomes Each dot represents an intermediate; each line represents an enzyme reaction.

4 Towards Bioreactors: The Cell Essentially, the cell can be seen as the smallest bioreactor (although these cell cultures are then again put into a reactor where they grow and produce the desired product). Cells are between ~ 0.1 μm (for some bacteria) and ~ 20 μm (human cells) in diameter. (Human are composed of about 10 trillion cells!) Remarkably, all cells are very similar in their structure even between plant cells and animal cells! From a chemical point of view, cells are essentially a self-replicating collection of catalysts (i.e. enzymes), or: a self-replicating catalytic reactor! L31-4

5 Cells & Bioreactors Beyond pure enzyme, many biochemical processes use living cells to catalyze a particular reaction. (In some way, this is a reactor in a reactor ) Because of the overwhelming complexity of the reaction pathways, and the fact that these pathways are almost always highly interconnected (i.e. you can rarely run only one clearly defined reaction pathway without engaging some side-reactions), clean mass balances in biochemical reactions are very hard to attain. Therefore, usually a simple balance over the total biomass in the system is taken. Biomass is thereby a term that refers to the sum of the cell cultures in the system. The generic reaction equation for a (here: aerobic) cell culture is: L31-5

6 Cell Growth L31-6 LAG PHASE stationary decline Log(N) log inoculate lag time EXPONENTIAL (LOG) PHASE Cells reach maximum rate of cell division (while nutrients and a environment are favorable) STATIONARY PHASE 1. Population reaches maximum numbers, rate of cell inhibition (death) = Rate of multiplication 2. DEATH PHASE 1. Decline in growth rate due to depletion of nutrients or death h due to toxic by-products 2.

7 Stoichiometry of Cell Growth Due to the complex net of interconnected reactions, the stoichiometry is difficult to describe precisely. Therefore, a lumped description is often used: cells + substrate -> more cells + product or, in a pseudo-chemical way: S > cells Y C/S C + Y P/S P where we introduced the yield coefficients: Part of the substrate is being consumed to maintain the cell s other activities (i.e. sustain the cell s life). This is captured by: L31-7 A typical value for m is: m = 0.05 (g substrate)/(g dry weight) h -1

8 Kinetics of Cell Growth The exponential growth of cells is often described by the Monod equation. where r g denotes the cell growth rate [g/(l s)], C C the cell concentration [g/l], and μ denotes the specific growth rate. μ can be expressed by: where μ max denotes the maximum specific growth rate [s -1 ], C S the substrate concentration [g/l] and K S is the Monod constant [g/l]. Hence, cell growth can be described (by combining the two equations above) by: L31-8 In many cases, K S is small, so that the rate simplifies to: r g = μ max C C

9 Product Inhibition & Cell Death L31-9 Since often a product of an enzymatic reaction act as a regulator to the reaction, i.e. it inhibits the further growth of the cells (remember the example of wine making fermentation of glucose to ethanol where > 18% alcohol is toxic to the cells?), this also needs to be accounted for in the kinetic equation. The specific form in which this accounted for depends on the individual system. A fairly common form is: with where C P denotes the product concentration and C P* the toxic concentration limit. (Obviously, despite the nomenclature, k app is not truly a rate coefficient!) The kinetics of the cell death is typically described by a simple rate law: where k d is the rate constant for the natural death of the cell, and k t the rate of death due to a toxic substance (with concentration C T ). Due to the exponential growth kinetics of cells, doubling times are often used to describe the kinetics. Typical doubling times range from 10 min to 2 hours for most cells cultures.

10 Mass Balance Equations L Just like for any reactor, we need to put stoichiometry and kinetics together to obtain complete mass balances in a bioreactor. Again, we cannot account for reactants/products in the form we are used to. Rather we need to balance either the number of cells in the system or the total (bio)mass in the system. Let s do the latter for a bio-cstr : rate of accumulation (of cells) [g/s] rate of inflow (of cells) [g/s] rate of outflow (of cells) [g/s] = + + and the corresponding balance for the substrate: rate of generation (of cells) [g/s] where the rate of substrate consumption in the Growth Phase is described by: r S = Y S/C (-r g ) - m C C (note that this is Y S/C, not Y C/S, i.e. the inverse of how we defined it above!) and the rate of substrate consumption in the Stationary Phase is described by: r S = Y S/P (-r P ) - m C C Note that typically the stationary phase is where the (bulk of the) product is formed!

11 Example: Bacteria Growth in BR So, let s put this all together: Calculate the time-dependent concentrations during the fermentation of glucose to ethanol in a batch fermentor. The fermentation is done by yeast (Saccharomyces cerevisiae yes, we are brewing beer!!) with an initial cell concentration of 1 g/l and a glucose concentration of 250 g/l. Additional data: parameters for Monod equation: μ max = 0.33 h - 1, K S = 1.7 g/l inhibition of cell growth by ethanol: C P* = 93 g/l, n= 0.52 Cell death coefficient: k d = 0.01 h -1 cell maintenance m= 0.03 (g ethanol)/(g cells h) Yields: Y C/S = 0.08 g/g,, Y P/S = 0.45 g/g L

12 The Equations Mass Mass Balances Balances Rate Rate laws laws cells cells (yeast): (yeast): V dc C /dt = (r g -r d ) V rr d = d = k d C C substrate substrate (glucose): (glucose): V dc S /dt = r S V rr S = S = -r g Y S/C m C C = -r g /Y C/S m C C product product (ethanol): 0.52 (ethanol): V dc P /dt = r g V Y P/C C P μmax C C r g = C S 1 * CP KS + CS Solve with numerical solver (here: Matlab)! L

13 Biochemical Reactors: The Fermenter Almost always CSTR or BR Often operated as semi-batch Exit Gas Flow Fresh Media Feed Level Sensor ph Sensor Dissolve O 2 Sensor Thermocouple Acid/base Antifoam Inlet Air Flow Agitator Sparser L Exit Liquid Flow

14 L Why are these reactors all so clean? Fermenters V > 10,000 L V > 10,000 L V > 10,000 L images:

15 Some Problems with Mixing Agitation Aeration In fermentation, additional consideration must be taken when designing mixers: at low aeration rate, oxygen supply becomes limiting at high aeration rates, undesired foaming occurs at low agitation, mixing is poor at high agitation, the shear forces can damage the (highly sensitive) cells L towards higher areation & agitation rates, cost can become an additional issue

16 Bubble Columns & Loop Reactors For aerobic fermenters, the mechanical stirrer (and the associated problems with mechanical damage to cells) can be replaced through mixing based on the bubbly gas flow in so-called bubble-column reactors and loop reactors. gas out gas in gas out gas in L bubble column loop reactor