 Microbial Cell Factories Tânia Sousa with contributions from :Bas Kooijman Gonçalo Marques

  Metabolism at the chemical level is very complex  “Knowledge on motors of cars is of little help to solve queuing problems” Using thousands of chemical species and chemical reactions to define the organism

  What is metabolism at a more aggregated level?  Using resources (energy and materials) to growth, to repair, to maintain the level of complexity and to reproduce  Focus: 1) mass and energy conservation; 2) full-life cycle; 3) dependence on the environment Metabolism at an aggregated level

 Applications

  Food Safety  How to prevent harmful micoorganisms from poisoning food? Applications salmonella Temperature range: 6-46 o C Optimum Temperature: 37 o C pH range: Optimum pH:

  Food Production  What are the optimal conditions for beer production? Applications

  Waste Water Treatment Plant  What are the necessary conditions to mantain an healthy microbian comunity in the biological reactors? Applications

  Two aspects of shape are relevant for energetics: surface areas and volume. Why? Metabolism: the importance of size

  Two aspects of shape are relevant for energetics: surface areas (acquisition processes) and volume (maintenance processes). Metabolism: the importance of size

  How does A/V change? Metabolism: the importance of size

  How does A/V change? Metabolism: the importance of size

 Diameter = 0.75  m Area = 0.44  m 2 Volume (with vacuole)= 0.22  m 3 Diameter =  m Area = 0.25  m 2 Volume = 0.3  m 3 A/V = m -1 Diameter = 0.2  m Area = 0.31  m 2 Volume = 0.42  m 3 A/V = m -1

 Metabolism: the importance of size

  Metabolism (respiration or heat production) as a function of mass  Metabolism increases with weight raised to the power 3/4  Max Kleiber originally formulated this basic relationship back in the 1930s. Kleiber’s Law: the importance of size

  The cyanobacterial colony Merismopedia – V1 morph  Colony is one cell layer thick  What happens to the relationship between A/V as the organism (colony) grows from L 1 to L 2 =2L 1 ? Metabolism: the importance of shape L1L1 L2L2

  The cyanobacterial colony Merismopedia – V1 morph  Colony is one cell layer thick  V1 morph (A grows proportional to V) Metabolism: the importance of shape L1L1 L2L2

  The cyanobacterial colony Merismopedia – V1 morph  Colony is one cell layer thick  V1 morph (A  V) Metabolism: the importance of shape L1L1 L2L2 V1 morph When the organism grows: acquisition and maintenance processes grow proportionaly

  Dynoflagellate Ceratium (marine phytoplancton)  Rigid cell wall that does not grow (internal growth at the expense of vacuoles)  What happens to the relationship between A/V as the organism (colony) grows from V 1 to V 2 ? Metabolism: the importance of shape

  Dynoflagellate Ceratium (marine phytoplancton)  Rigid cell wall that does not grow (internal growth at the expense of vacuoles)  V0 morph (A is constant) Metabolism: the importance of shape

  Dynoflagellate Ceratium (marine phytoplancton)  Rigid cell wall that does not grow (internal growth at the expense of vacuoles)  V0 morph (A is constant) Metabolism: the importance of shape V0 morph When the organism grows: acquisition remains constant but maintenance grows

  Several Archaebacteria (spheres)  All linear body dimensions scale up or down by the same multiplier  What happens to the relationship between A/V as the organism (colony) grows from D 1 to D 2 =2D 1 ? Metabolism: the importance of shape

  Several Archaebacteria  All linear body dimensions scale up or down by the same multiplier  Isomorph (A  V 2/3 ) Metabolism: the importance of shape

  Several Archaebacteria  All linear body dimensions scale up or down by the same multiplier  Isomorph (A  V 2/3 ) Metabolism: the importance of shape Isomorphy When the organism grows: maintenance grows faster than acquisition

  Isomorph: surface area proportional to volume 2/3  V0-morph: surface area proportional to volume 0  the dinoflagelate Ceratium with a rigid cell wall  V1-morph: surface area proportional to volume 1  The cyanobacterial colony Merismopedia Metabolism: the importance of shape

  Population behaves as a super V1-organism Shape: the population level

  What defines an organism?  M v - Mass of Structure  M E - Mass of Reserve Organism: the State Variables M – molar mass (C_moles) V – indice for the compound of structure E – indice for the compound reserve E Hopf Courtesy of Jan Heuschele and Starrlight Augustine V

  Strong homeostasis  Reserve & structure have constant aggregated chemical composition Strong homeostasis

  Strong homeostasis  Reserve & structure have constant aggregated chemical composition Reserve & Structure: Strong homeostasis  Why more than 1 state variable to define the biomass?  The aggregated chemical composition of organisms is not constant – it changes with the growth rate  Why not more than 2 state variables to define biomass?  Two are typically sufficient (in animals and bacteria) to capture the change in aggregated chemical composition with the growth rate What does a variable aggregated chemical composition implies?

  Weak homeostasis  At constant food level organisms tend to constant aggregated chemical composition What has to be the relationship between M V and M E to ensure a constant aggregated chemical composition? Reserve & Structure: Weak homeostasis

  The boundary of the organism  Rectangles are state variables Organism: mass & energy description M E - Reserve M V - Structure  Chemical and thermodynamic properties of the structure and reserve are constant (strong homeostasis)

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: feeding & assimilation M E - Reserve M V - Structure Feeding Assimilation Notation X - Food A – Assimilation dots – per unit of time - molar flow of compound X in transformation A

  Feeding: the uptake of food  Assimilation: conversion of substrate (food, nutrients, light) into reserve(s)  Depend on substrate availability & structural surface area Feeding & Assimilation  Mass transfer (needed for acquisition and food processing) is proportional to area  Strong homeostasis imposes a fixed conversion efficiency

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: feeding & assimilation M E - Reserve M V - Structure Feeding Assimilation

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: feeding & assimilation M E - Reserve M V - Structure Feeding Assimilation How do we obtain the energy description?

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: feeding & assimilation M E - Reserve M V - Structure Feeding Assimilation

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: mobilization M E - Reserve M V - Structure Feeding Assimilation Mobilization Notation C - Mobilization

  Microorganisms are capable of spending energy on growth in the absence of food  Mobilization from reserve -> higher control over metabolism (independence from the environment) Mobilization of Reserve

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: mobilization M E - Reserve M V - Structure Feeding Assimilation Mobilization energy description

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes Organism: mobilization M E - Reserve M V - Structure Feeding Assimilation Mobilization energy description

 Priority maintenance rule

  The priority maintenance rule states that maintenance has priority: from maintenance is paid first and the rest goes to growth Priority maintenance rule  The priority maintenance rule results from the demand driven behavior of maintenance

  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circle is the priority maintenance rule. Organism: maintenance M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance

  Collection of processes that maintain the organism alive:  protein turnover (synthesis, but no net synthesis)  maintaining conc. gradients across membranes  (some) product formation  movement Somatic maintenance

  Reserve compounds have no maintenance needs because they have a limited lifetime Somatic maintenance M E - Reserve Assimilation Mobilization

  Reserve compounds have no maintenance needs because they have a limited lifetime  Somatic maintenance:   structural volume (most costs)   surface area: osmoregulation in bacteria Somatic maintenance  Specific somatic maintenance costs are constant because the chemical and thermodynamic properties of the structure are constant (strong homeostasis)

  Metabolism in a DEB individual.  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circles is the priority maintenance rule. Organism: maintenance M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance

  Metabolism in a DEB individual.  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circles is the priority maintenance rule. Organism: maintenance M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance Energy description

  Metabolism in a DEB individual.  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circles is the priority maintenance rule. Organism: growth M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance Growth

  Growth is the increase of the amount of structure (net synthesis of protein)  Allocation to growth (supply driven): Growth

  Growth is the increase of the amount of structure (net synthesis of protein)  Allocation to growth (supply driven): Growth What is the difference between these two fluxes? Why is y VE a parameter?  Strong homeostasis imposes a fixed conversion efficiency

  Growth is the increase of the amount of structure (net synthesis of protein)  Allocation to growth (supply driven): Growth Write the energy description

  Growth is the increase of the amount of structure (net synthesis of protein)  Allocation to growth (supply driven): Growth Write the energy description

  Metabolism in a DEB individual.  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circles is the priority maintenance rule. Organism: metabolic processes M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance Growth

  What are the dynamics of the state-variables? Organism Dynamics

  The dynamics of the state-variables are given by: DEB Dynamics

  The dynamics of the state-variables are given by: Meaning [E G ]? DEB Dynamics [E G ]- specific costs of growth

 Dynamics of reserve density

  What happens at constant substrate level?  What is the maximum level of reserve density? Dynamics of reserve density

  What happens at constant substrate level? dm E /dt=0  What is the maximum level of reserve density?  What is the value for m E as a function of m Em in weak homeostasis? Dynamics of reserve density

  What happens at constant substrate level?  What is the maximum level of reserve density?  What is the value for m E as a function of m Em in weak homeostasis? Dynamics of reserve density dm E /dt=0

 Weak homeostasis

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  Microbes: have a variable temperature mostly dictated by environmental conditions  Bacteria are classified by their optimal growth temperature: Metabolic rates: the effect of temperature Methanopyrus kandleri (122ºC)

  How do metabolic rates ln k ( T ) depend on temperature? ln rate Metabolic rates: the effect of temperature

  How do metabolic rates depend on temperature? ln rate Metabolic rates: the effect of temperature

  How do metabolic rates depend on temperature? Metabolic rates: the effect of temperature

  How do metabolic rates depend on temperature?  What is the meaning of the slope cte= T A ? Metabolic rates: the effect of temperature

  How do metabolic rates depend on temperature?  What is the meaning of the slope cte= T A ?  Write the constant as a function of T 1  Write na expression for k(T) Metabolic rates: the effect of temperature

  The Arrhenius relationship has good empirical support  The Arrhenius temperature is given by minus the slope: the higher the Arrhenius temperature the more sensitive organisms are to changes in temperature  Arrhenius relationship:

 Metabolic rates: temperature range

  The Arrhenius relationship is valid in the temperature tolerance range  At temperatures too high the rates fall abruptly to zero  At temperatures too low the rates are usually lower than predicted by the Arrhenius relationship Metabolic rates: temperature range

 Metabolic rates: the effect of temperature  All metabolic rates depend on temperature on the same way because otherwise it would be difficult for organisms to cope with changes in temperature (evolutionary principle)

  All parameters that have units time -1 depend on temperature Metabolic rates: the effect of temperature

  How does the energy flow rate that is used for maintenance needs depends on temperature? Metabolic rates: the effect of temperature

  Metabolism in a DEB individual.  Rectangles are state variables  Arrows are flows of food J XA, reserve J EA, J EC, J ES, J EG or structure J VG.  Circles are processes  The full circles is the priority maintenance rule. A DEB organism Assimilation, dissipation and growth M E - Reserve M V - Structure Feeding Assimilation Mobilization Maintenance Growth

  Assimilation : X(substrate)+M  E(reserve) + M + P  Dissipation : E(reserve) +M  M  Growth : E(reserve)+M  V(structure) + M  Compounds :  Organic compounds: V, E, X and P  Mineral compounds: CO 2, H 2 O, O 2 and N waste 3 types of aggregated chemical transformations

 E - Reserve V - Structure Catabolism: Maintenance:Growth: Assimilation: Klebsiella Aerogenes  Characteristics: Gram-negative bacteria and a facultatively anaerobic rod (V1-morph). T=35ºC pH: 6.8 O 2, NH 3 X – Glycerol C 3 H 8 O 3 CO 2, H 2 O, and sensible heat Biomass: E+ V CH 1.64 O N CH 1.66 O N 0.312

  Obtain the aggregated chemical reactions for assimilation, dissipation and growth for klebsiella aerogenes in a chemostat  Identify in these equations y XE, y PE and y VE.  Constraints on the yield coeficients  Degrees of freedom Exercises

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  The stoichiometry of the aggregate chemical transformation that describes the organism has 3 degrees of freedom: any flow produced or consumed in the organism is a weighted average of any three other flows

  Write the energy balance for each chemical reactor (assimilation, dissipation and growth) Exercises

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  Indirect calorimetry (estimating heat production without measuring it): Dissipating heat is weighted sum of three mass flows: CO 2, O 2 and nitrogeneous waste (Lavoisier in the XVIII century).

 Dissipating heat Steam from a heap of moist Prunus serotina litter illustrates metabolic heat production by aerobic bacteria, Actinomycetes, fungi and other organisms

 E - Reserve V - Structure Catabolism: Maintenance:Growth: Assimilation: Klebsiella Aerogenes in DEB Theory  Characteristics: Gram-negative bacteria and a facultatively anaerobic rod (V1-morph). T=35ºC pH: 6.8 O 2, NH 3 X – Glycerol C 3 H 8 O 3 CO 2, H 2 O, and sensible heat Biomass: E+ V CH 1.64 O N Reserve Turnover Rate:  E =2.11h -1 CH 1.66 O N y XE =1.345 y VE =0.904  M =0.021h -1 Maintenance Rate Coefficient: Energy Investment Ratio: g=1

D(h - 1) Measurements (points) and DEB model results (lines). Comparison with experimental data I yield (C-mol Woutput.C-mol X -1 ) O 2 (mol O2.C-mol Woutput - 1.h -1 ) CO 2 (mol CO2.C-mol Woutput -1.h - 1 ) Esener et al. (1982, 1983)

Measurements (points) and DEB model results (lines). Comparison with experimental data II n HW (mol H.C-mol W -1 ) n OW (mol O.C-mol W -1 ) n NW (mol N.C-mol W -1 ) Esener et al. (1982, 1983) D(h - 1)

Heat Production vs. Dilution rates kJ per mol O 2 consumed kJ per C-mol biomass inside the chemostat per hour kJ per C-mol biomass formed Thornton’s coefficient D(h - 1) Irreversibilities are equal to the amount of heat released Production of biomass becomes more efficient

Measurements (points) and DEB model results (lines). Comparison with experimental data II n HW (mol H.C-mol W -1 ) n OW (mol O.C-mol W -1 ) n NW (mol N.C-mol W -1 ) Esener et al. (1982, 1983) D(h - 1)

Heat Production vs. Dilution rates kJ per mol O 2 consumed kJ per C-mol biomass inside the chemostat per hour kJ per C-mol biomass formed Thornton’s coefficient D(h - 1) Irreversibilities are equal to the amount of heat released Production of biomass becomes more efficient