Transcript Lecture 2 - Portal UniMAP

Simulation Of Bioprocess
ERT 315/4
TYPES OF BIOPROCESS
AND
BIOPRODUCTS
Criteria to select appropriate
biocatalyst
 What yield, product concentration, and productivity can be reached?
 What substrate can be utilized, what additional media components
are required, and how does it all effect downstream processing?
 What by-products are formed and how do they affect yield and
downstream processing?
 What are the challenges in biocatalyst preparation, storage,
propagation, security, and safety?
 What are the optimal reaction conditions, e.g. temperature, oxygen
supply, shear sensitivity, foam formation, etc?
 How well do we understand the reaction mechanisms, are they
robust and genetically stable?
 If the product is expressed intracellularly, how is it extracted?
 How do we purify the desired product form the many impurities in
the process?
Characteristics of biocatalyst
Biocatalyst
Production
device
Raw
material
Timescale
Purification
Complex protein
structure
Viral/prion
risk
Process
examples
Enzymes
Bioreactor
Pure substrates
Short
Simple
No
No
Simple media
Short
Medium
No
Small
Bioreactor
Bioreactor
Simple media
Complex media
Medium
Medium
Medium
Medium
No
Yes
Small
Medium
Plant cells
Bioreactor
Simple media
Medium
Medium
Possible
Small
Transgenic
plants
Bioreactor
Fertilizer, CO2,
various others
Long
Complex
Possible
Small
Transgenic
animals
Whole plant
Various plant &
animal materials
Certain parts of
plants, animals
and humans
Long
Complex
Yes
High
Long
Complex
Possible
High
Cyclodextrin,
Acrlyamide, L-dopa
Lysine,Vitamin B2,
Insulin
Citric acid, antibiotics
Monoclonal
Antibodies,
Interferons
Taxol, shikonin,
Methyldigoxin
Antibodies, antibody
Fragments, HAS,
PHB
Α1-antirypsin, HAS,
Lactoferrin
Plasma,components,t
axol
Bacteria and
yeast
Fungi
Mammalian
cells
Bioreactor
Extractive
technology
Whole animal
Biocatalyst
Enzyme Biotransformation
-enzymes: protein with a unique three-dimensional structure able
to bind a substrate, usually but not always a small molecule, and
catalyze a specific reaction, similar to chemical catalysis but under
mild conditions of temperature and pressure
-classified in six groups according to the chemical reaction: oxidoreductase, transferases, hydrolases, lyases, isomerases, ligases
-highly selective and specific in the reaction: regio-, stereo- and
enantioselective
-can be in solution or immobilized
-five major of reactions where enzymes are used industrially:
hydrolysis of proteins, palysaccharides,esters,amides, nitriles,
epoxides; synthesis of esters, amides, glycosides; carbon-carbon
bond formation; reduction reaction; oxidation reactions
Metabolic Bioconversion using cell cultivation
- Classified into five groups: Bacteria, Fungi, Mammalian cells,
insect cells, plant cells
(i) Bacteria
-unicellar prokaryotes with a rigid cell wall
-key determinants for their growth: media composition, temp.,
gaseous environment, pH
-show a range responses to oxygen: aerobic bacteria- require
oxygen for their growth; anaerobic bacteria- grow only at the
absence of oxygen; facultatively-anaerobic bacteria- able to grow
under both conditions
-temperature optimal for growth: psychrophiles (20-30 °C),
mesophiles (30-40 °C), thermophiles (45-60 °C), extreme
thermophiles (extremophiles) (80-105 °C)
-optimum pH: 6.5-7.5
(ii) Fungi
-divide into two subgroups: yeasts, molds
-yeast: small, single cells that can grow as individual cells or
clumps
-molds: multicellular, vegetative structure call mycelium, as
usually highly-branched systems of tubules
(iii) Mammalian cells
-produce correctly folded proteins and secrete them to the culture
environment
-grow quite slowly, with typical doubling times of 12-20 h
-temperature : 37 °C, pH: 7.3
-mammalian cell product: monoclonal antibodies, interferons,
vaccines, erythropoietin
(iv) Insect cells
-produce recombinant proteins less expensively and more quickly
than mammalian cells and at high expression levels
-typically grow at around 28 °C, and pH 6.2
-used for veterinary vaccines for the swine fever virus
(v) Plant cells
-10 to 100 times larger than microbial cells and more sensitive to
shear
-slow metabolism, with doubling times of 20-100h
-cultivate as a callus or a lump of undifferentiated plant tissue
growing on a solid nutrient medium or as aggregated plant cells in
suspension
-used to produce secondary metabolites, anticancer drug paclitexel
(taxol), recombinant proteins of high value
Transgenic Plants
-genetically modified plants to produce a wide variety of products
-the expression can take place in the whole plant or only in a
certain part as in the seeds
-commonly used plants: tobacco, potato, rice, wheat
-inexpensive, easy to scale-up, free of human pathogens
Transgenic Animals
-reduce the dependency on the seasonal and geographical
conditions, post-translational modifications are more likely to
mimic the native structure
-usually done by injecting exogenous DNA into egg cells to
produce a vital embryo that is later able to express the desired
product
Extractive Technologies
-comprise all processes where a product is extracted from
natural material
-Used in the extraction of pharmaceuticals from human or
animal blood and from plant material
-the products usually chemically complex non-protein materials
Bioproducts
Product Classifications/Characteristics
-according to size, bioproducts can be divided into:
Small molecules, Large molecules and Solid particles
-Small molecules
i. sugars, amino acids, organic acids, vitamins
ii. Molecular weight of 30-600 Da and a radius smaller than 1 nm
Iii divided into primary and secondary metabolites:
a. Primary metabolites: sugar, organic alcohols, acids-produced
in the primary growth face of the organism
b. Secondary metabolites: formed at or near the beginning of the
stationary phase, e.g. antibiotics and steroids
-large molecules
i. proteins, nucleic acids, polysaccharides
ii. Molecular weight of 103-106 Da
-solid particle
i. Whole cell like yeast, animal cell, ribosomes, viruses
ii. A radius of up to several µm
-by
the scale of production, bioproducts can be classified into:
Bulk or commodity chemicals made at large scale, Fine
chemicals and Pharmaceuticals made a smaller scale
-bulk chemicals
i. Produced in very large amounts
ii. Simple downstream processing
iii. Sold at a relatively low price
-pharmaceuticals
i. produced in a small amounts
ii. high price
iii. used expensive media and complex equipment with low
productivities
-fine chemicals
i. Used as intermediates and have application in a variety of
industries
ii. Annual production, price, and required purity lie between bulk
chemicals and pharmaceuticals
Product classes
-describe by its function (proteins, organic acids, lipid) or application
(food and feed additives, pharmaceuticals, detergents, chemical
intermediates, agriculturally used products)
i. organic alcohol and ketones
-produced in anaerobic fermentations
ii. organic acids:
-used as intermediates or as food additives
-major organic acids produced are citric, lactic, gluconic acid.
iii. amino acids:
-the building blocks of protein and are connected via peptide bonds
-used as food additives, feed additives, and in pharmaceuticals
iv. nucleic acids:
-used as therapeutics, e.g. DNA vaccines, gene therapy
v. antibiotics:
-frequent use in human and animal health
-produced on fungal fermentation
vi. vitamins:
-produced in bioprocesses, e.g. vitamin A, C, E, and the B vitamins
vii. biodegradable biopolymers:
-plastics derived from renewable material
-common biopolymer are polyhydroxyalkanoates (PHA)
viii. dextran and xanthan:
-industrially produced microbial polysaccharides
-used as thickening, gelatinizing, suspending agents
ix. carotenoids:
-natural pigments (yellow or red color)
-produced by microorganisms
x. pesticides
xi. lipids:
-including fats, oils, waxes, phospholipids, steroids
-commercially produced lipid: prostaglandins, leukotrienes,
xii. proteins
Raw materials
-Water:
-dominant raw material
-other component of the reaction medium: macronutrients and
micronutrients
-macronutrient:
-needed in concentrations larger than 10-4 M
-including carbon–energy source, oxygen, nitrogen, phosphate,
sulfur, and some minerals such as magnesium and potassium ions
-carbon-energy source:
-provides the carbon for biosynthesis as well as energy derived
by its oxidation
-Typically used carbons sources: glucose, starch, corn syrup,
molasses. soybean oil, palm oil, ethanol, methanol
-50% is incorporated in the biomass, and remaining 50% is used
to derived energy for biosynthesis
-nitrogen:
-accounts for 10-14% of the dry cell mass
-most widely used are ammonia and ammonia salts, proteins,
-oxygen and hydrogen:-20% of the cell mass (O2), 8% (H2)
-phosphorus: 3% of cell dry weight and is provided by phosphate
salts
-sulfate: 0.5% of cell mass is added as sulfate salts or with amino
acids contained in complex media
-magnesium and potassium ions: provided as inorganic potassium
and magnesium sulfate
-micronutrients:
-required in low concentrations:
-including iron, zinc, manganese,copper, sodium, calcium, boron
-added as inorganic salts
-also can be classified into defined or synthetic media and complex
or natural media
-defined media:
-contain specific amounts of pure chemicals with a known
composition
-complex media:
-include one or more natural materials whose chemical composition
is not exactly known and which may vary with source of time
-natural media:
-cheaper
-cause less reproducible fermentation and more complex
downstram processing
-bacteria and fungi: need only a relative simple media and very
low cost
-mammalian cells: more complex medium is necessary, need
serum as required ingredient
(complex media) or not (synthetic media)
-plant cell: require a carbohydrate cell source, inorganic
macronutrients and micronutrients.
1. Avoid waste formation
2. Reduce waste formation
Economic savings
3. Extend material use
4. Recycle material
Ecological costs
Waste treatment, Reduction and
Recycling
5. Downcycle material
6. Treat waste/energetic recovery
7. Safe water disposal
Steps of waste avoidance and treatment
1. Avoid waste formation
-to avoid the formation of waste
-if feasible and cost-effective, subsequent treatment is unnecessary
-if cannot be prevented completely, try to reduce it
2. Reduce waste formation
-reuse of material
3. Recycle material use
-recycling of an organic solvent used in an extraction step
-Compared the amount recycle and the amount of material and
energy necessary for the recycling to decide it is economically and
environmentally favorable
-if cannot be recycled because the purification becomes too
expensive, used another purpose that requires less purity
4. Treat waste/energetic recovery
-have to be treated or disposed safely
Types of waste
-gaseous stream:
-cause from the exhaust air from a bioreactor (contains air, CO2,
and water), distillation and evaporation steps
-solid waste:
-categorized as hazardous and non-hazardous waste
-hazardous waste:
-containing heavy metals or highly toxic substances
-need special treatment or disposal with high-safety mannerscause higher costs
-non-hazardous waste:
-wet biomass
-if a recombinant organism is used, sterilization of the material is
necessary, usually by heat
-liquid waste:
-treated in a biological sewage treatment plant at the production
site of the bioprocesses
Overall development process
Literature/patent review
Biocatalyst screening
Development steps
Biocatalyst optimization
Medium and reaction condition optimization
Selection of downstream steps
Identification of PFD
Optimization of unit operations
Plant size
Scale-up : Lab – technical - industrial
Approval, clinical trials
Process modeling and uncertainty analysis
Economic and environmental assessment
Development process
Product idea
Process
Raw
material
Upstream
processing
Consumables
Utilities
Labor
Bioreaction
Donwstream
processing
Waste
Treatment/
disposal
Final
product
Sustainability
Assessment
Sustainability Assessment
What is “sustainability”?
-the development that meets the needs of the present without compromising the
ability of the future generations to meet their own needs (Brundtland, 1980s)
-the optimal growth path that maintains economic development while protecting
the environment and optimizing the social conditions with the boundary of relying
on limited, exhaustible natural resources
-a business approach that creates a long-term shareholder value by embracing
opportunities and managing risks deriving from economic, environmental, and
social development
Sustainability
Economic Ecological
Social
Economic Assessment
Bioengineering
Conversion, yield
Raw materials
Process flow diagram
Volume/mass
of product
Equipment
prices
Utilities/waste
Purchase equipment
cost
Labor
Consumables
Operating cost
Multipliers
Capital investment
1.Estimate the capital investment
2.Operating costs can be derived from the different cost items
Capital cost estimation
-total amount of money that has to be spent to supply the necessary plant (the
fixed capital investment) plus the working capital that is needed for the operation
of the facility
-how to estimate:
i. Equipment purchase cost
ii. Estimation of total capital investment:
a. Direct cost- the purchased equipment that need to be installed
b. Indirect cost- a number of planning costs, like the preparation of design books
that document the process, the design of equipment, etc
iii. Multiplier values –for a realistic estimation of the capital investment, derived
from the empirical data and are different for different process types
iv. Prices indices- changes of equipment price over time due to inflation/deflation
or market conditions
Present prices = Price at t0 x index value today/ index value at t0
v. Scale-up Factors- the cost of a single piece of equipment or a complete plant
changes when its capacity is changed
C2= C1 x (q2/q1)0.6, where C1: coast of a plant with a certain capacity q1
Operating-cost simulation
-the total of all costs operating the plant and recovering the capital investment,
i.e. the annual amount of money necessary to produce the product and pay back
investment cost
-Divided into variable, fixed, plant overhead cost
-Variable cost:
1. Raw materials – the list of raw materials and the amounts consumed
2. Consumables- all material and equipment parts that have to be replaced from
time to time
3. Labor- determined by the operator hours and the hourly wage
4. Operating supplies- including clothing, tools, and protective devices for the
workers and also everyday items needed to run the plant
5. Laboratory, quality control, and quality assurance
6. Utilities- energy consumption for heating, cooling, evaporation/distillation,
aeration, etc
5. Waste treatment and disposal
6. Royalty expenses- single unit operations or even the whole process that has
to be covered by a patent owned by others
-Fixed Cost (Facility-dependent cost)
1. depreciation- a capital investment that was need to built a plant and this
investment has to be paid by charging an annual amount of money
2. Maintenance and repair
3. Insurance and local taxes-derived from the direct fixed capital (DFC)
4. Rent and interests
-Plant overhead cost –factory expenses, or plant overhead costs caused by the
operation of facilities that are not directly related to the process, e.g. medical
service, safe and protection
Others:
-general expenses- the general expenses that has to cover to manage the
company, to sell product and computer support. Including administration,
distribution and marketing, research and development
-Unit production cost (UPC)- the total product cost allocated to the annual
amount of product
Profitability Assessment
-revenues: sum of all sales of the main and side product of a process within a
certain time period usually a year. For a single-product facility, the revenue r
for year j is :
rj= mjxpj ,
where mj is the amount of product sold in year j and pj the (average) price
realized in this year
-measurement of profitability- a number of indices that are used to evaluate the
profitability of a process
i. The gross profit in year j (Gj) is the annual revenue rj minus the annual total
product cost including depreciation:
Gj=rj-cj
ii. The net profit in year j (Nj) is the gross profit minus the income tax. The
income tax is determined by the tax rate Ф
Nj = (rj-cj)x(1- Ф) = Gjx(1- Ф)
iii. The net cash flow in year j (Aj) is the sum of net profit and the depreciation dj
of the year
Aj=Nj+dj
n
j+1
iv. The return on investment (ROI) is the ratio of profit to investment and
measures how effectively the company uses its invented capital to generate
profit. Usually calculated using the net profit and the total investment (TCI) and
is shown in percentage value:
ROI=Nj/TCI x 100
v. The payback period (PBP) is the length of time necessary to pay out the
capital investment by using the annual cash flow that return to the company’s
capital reservoir. In most cases, the direct fixed capital (DFC) is used for this
index
PBP=DFC/Aj
The PBP also can be calculated using the TCI and the net profit
PBP=TCI/Nj=100/ROI
Time value of money: time-value of the earned money
NPV=ΣAj/(1+i)j
Environmental Assessment
-to identify the environmental ‘hot spots’ of the process
Process characteristics
Modeling and simulation
Component properties
Impact categories
Material balance
ABC classification
Mass indices (MI)
Environmental factors
(EF)
Environmental indices
(EI)
Impact categories
Process
Components
Assessment structure of the method
Structure of the method
-two starting point:
a. Process and characteristics by Superpro Designer model
-the result is the material balance of the process
-from the material balance, Mass Index (MI) can be calculated for all input
(states how much of component is consumed to produce a unit amount of the
final product) and output components (how much of a component is formed per
unit final product)
b. From the component properties
-classified into A, B and C classes that represent environmental relevance
(high, medium, low relevance) from the 15 impact categories (IC) that may have
a negative effect on human health and the environment
-the IC are allocated to six impact groups, representing an important field
Concerning environmental, health or safety aspects
-numerical values for the classes A, B and C, and a weighting factor
(=environmental factor) is derived from its classifications in the impact groups
-next step:
-Link the amount of the components in the mass balance with Environmental
Factors
-The resulting Environmental Index (EI) helps to identify those components that
are environmentally most relevant in the process
Weighting factors/indices
Calculation
Mass Index component i, MIi (kg/kg P)
mi= amount of component i (kg);
mp= amount of final product (kg P)
MI=mi/mp
Mass Index process, MIprocess (kg/kg P)
MIprocess,In =Σmi/mp
MIprocess,Out=1+Σmi/mp
EFMv,i=(IG1,i+IG2,i+IG3,i+IG4,i)/j
Environmental Factor component i,
EFi (index points/kg)
Via arithmetic average; as EFMv,i,In/EFMV,i,Out
IGj,i= value of component i in Impact Group j;
j= Number of impact Groups
Environmental Factor component i,
EFi (index points/kg)
Via multiplicatiom; as EFMult,i,In and EFMult,i,Out
EFMult,i=πIGj,i
Environmental Index component i,
EIi (index points/kg P) (as EIi,In or EIi,out)
EIi=EFi,x mi/mp=EFi x MIi
Enviromental Index process
EIprocess (index points/kg P) (as EIIn or EIOut)
EIprocess=ΣEIi
Generak Effect Index Process
GEI (nondimensional)
GEI=EIprocess/MIprocess
Impact categories
Impact groups
Environmental Factors
Raw Material Availability
Land Use
Resources
Complexity of Synthesis
Grey Input
Thermal Risks
Component Risk
Environmental Factor
Input component
Acute Toxicity
Chronic Toxicity
Organism
Ecotoxicity
Global Warming Potential
Ozone Depletion Potential
Acidification Potential
Air
Photochemical Ozone Creation Potential
Environmental Factor
Output component
Odor
Eutrohication Potential
Water/Soil
Organic Carbon Pollution Potential
Impact category
I/O
Class A
Raw Material
Availability
I
Only fossil,
Only fossil, predicted
Predicted exhaustion exhaustion in 30-100
years
within 30 years
Land use
Critical material
used
I
I
Complexity of the
Synthesis
Thermal risk
I
I/O
>100m2/kg
Critical materials like
heavy metals, AOX,
PCB used or
produced in
stoichiometric
amounts
>10stages
R 1-4, 9, 12, 15-17,
44; EU; F+, E; NFPA
F+R: 3,4
EU:T+; R 26-28,32;
CH-poison class:
1,2; NFPA H:4; WGK
3;
ERPG:<100mg/m3;
IDLH: <100mg/m3
Acute toxicity
I/O
Class B
>10m2/kg and<100m2/kg
Critilcal materials involved
in sub-stochimetric
amounts
Class C
Exclusively
renewable, or
guaranteed long term
supply (>100 years)
<10m2/kg
No critical
compounds involved
3-10stages
<3 stages
R 5-8, 10, 11,14,18,19, 30; NFPA F+R: 0,1
EU: F, O; NFPA F+R:2
EU: T, Xn,Xi,C; R 20-25,
29, 31,34-39,4143,65,66,67; NFPA H: 2,3;
WGK 2; ERPG: 1001000mg/m3; IDLH: 1001000 mg/m3
CH-poison class: 5;
NFPA H: 0,1; WGK 1,
ERPG: >1000mg/m3;
IDLH:>1000mg/m3
Impact category
I/O
Class A
Class B
Class C
Chronic Toxicity
I/O
MAK:1mg/m3;
IARC;1,2A;R 4549,60,61
MAK:>10mg/m#;IAR
C: 4: CH-poison
class: 3,4,5
Ecotoxicity
I/O
EU:N;R 50 WGK 3;
MAK:110mg/m3;IARC;2B,3; R
33,40,62,63; EU: T,T+,Xn;
CH-poison class: 1,2
R 51-58; WGK 2
Global Warning
Potential
Ozone depletion
potential
Acidification
potential
Photochemical
ozone creation
potential
Odor
O
GWP>20
GWP<20
O
ODP>0.5
ODP<0.5
O
AP>0.5
AP<0.5
O
POCP>30 or NOx
30>POCP>2
Euthrophication
potential
Organic carbon
pollution potential
O
O
O
Odor threshold
<300mg/m3
N-content>0.2 or Pcontent>0.05
N-content<0.2 and Pcontent<0.05
ThOD>0.2 g O2/g
substrate
WGK ! Or no water
hazard
No global warming
potential
No ozone depletion
potential
No acidification
potential
POCP<@ or no
effect known
Odor threshold
>300mg/m3 or no
odor compound
without N and P
Compound without N
and P
ThOD<0.2g O2/g
substrate or no
organic compound
Social Assessment
Aspect
Health and safety
Quality of working conditions
Employment
Education and training
Social Indicators
Technology development
Technology application
-risk group of biological
substances
-risk factors for health and safety
-voluntary health measures
-quality of health and safety
management
-working time arrangement
-degree of psychological strain
-Percentage of women in leading
positions
-measures taken to improve
working conditions
-safeguarding of jobs
-continuity of job creation effects
-regions of job creation
-extent of job creation
-focus on employee training
-quality of human resource
management
-identification of training needs
-incorporation of employee
expectations
-job security levels
-amount of hazardous substances
-voluntary health measures during
application
-voluntary health measures during
usage
-working time arrangements
-degree of psychological strain
-percentage of women in leading
positions
-measures taken to improve working
conditions
-safeguarding of jobs
-continuity of job creation effects
-regions of job creation
-extent of job creation
-apprenticeship
-voluntary training offerings
-identification of training needs
-incorporation of employee expectations
Aspect
Knowledge management
Innovation potential
Product acceptance and social
benefit
Societal dialogue
Social Indicators
Technology development
Technology application
-Degree of knowledge exchange
-Used information system
-Control of knowledge exchange
-Employee involvement in
decision-making
-commercial exploitation potential
-contribution to scientific debate
-management of patents and
licenses
-number and types of patents
-stakeholder involvement
-usage of genetic engineering
methods
-social standards in supply chain
-societal benefits
-voluntary provision of information
-reporting of core activities to
neighborhood
-stakeholder involvement in
strategic decision making
-communication channels to
political debates
-aspects of knowledge exchange
-Used information system
-Control of knowledge exchange
-Employee involvement in decisionmaking
-degree of innovation
-product readiness and marketability
-estimated market penetration
-number and types of patents
-product acceptance
-usage of genetic engineering methods
-social standards in supply chain
-societal benefits
-used communication channels
-reporting of core activities to neighbors
-targeted dialogue partners
-measures taken to promote dialogue