Industrial biotechnology

Key technology in a bio-based economy

Kristel Bernaerts
Department of Chemical Engineering, KU Leuven
July 2014, BEST Summer Course

Outline
Setting the scene: a bio-based economy
What is industrial biotechnology
Fermentation process
Stages, equipment, techniques, costs, etc.
Sustainability of IB-based products

Bio-based economy
The application of biotechnology for the sustainable processing
and production of chemicals, materials and fuels from biomass
creates an opportunity to reduce significantly our dependence on
coal, oil and gas.
Biomass is an organic renewable energy source that includes
materials such as agriculture and forest residues, energy crops,
and algae.

Building a Bio-based Economy for Europe in 2020 (EuropaBio, policy guide)

Expectations of a bio-based economy

Society that is far less dependent on fossil fuels for energy
and industrial raw materials.
Greater use of renewable and expandable resources offers
increased potential for cutting greenhouse gas emissions.
Production processes that use less water and energy and
generate less waste can make industry both more sustainable
and more competitive.
Creation of new non-food markets for crops, together with
the emergence of alternative income sources for farmers, can
give depressed rural areas a new lease of life.
Building a Bio-based Economy for Europe in 2020 (EuropaBio, policy guide)

Source: US Department of Energy

from fossil to bio--based economy:

typical fossil product tree

from fossil to bio-based economy:

bio-based product tree

INDUSTRIAL BIOTECHNOLOGY

Key technology in a bio-based economy

Industrial (White) biotechnology

Application of biotechnology for industrial purposes.
Use of micro-organisms (fungi, bacteria) or parts of them
(enzymes) to produce industrially useful products.

Cell itself
Metabolite
Recombinant product

Use of micro-organisms:
historical background
Since ancient history (Egypt, Babylon, ...)
Fermented foods
Beverages: wine, beer, milk, ...
Foods: vegetables, yoghurt,
cheese, bread, ...
Microbial process:
Sugar(s) > alcohol(s)
Sugar(s) > organic acids
Application:
Food preservation
Create desired food properties

Illustration of brewing in Ancient

Egypt in the time of the Pharaohs

Use of micro-organisms:
historical background
Around 1860: discovery of the causative agents of alcoholic and
lactic acid fermentation (Louis Pasteur).
Around 1880: Bakers yeast, an independent yeast industry started
to develop alongside the distillery industry.
1914 to 1918: Large-scale glycerine production by fermentation
processes, organized by the German government to meet the
military needs for nitroglycerine.
1928-1929: discovery of first antibioticum by Alexander Fleming.
Penicilline produced by Penicillium chrysogenum. During WO
II, USA started with large-scale production of Penicilline.

Use of micro-organisms:
historical background
Since 1945 ...
... due to major technological developments (e.g., sterile
fermentation, aerobic processes) and driven by chemical
pharmaceutical industry (antibiotics) ...
... due to development of recombinant DNA technology (1975 - )
Era of modern fermentation technology and biotechnology
due to advances in metabolic engineering, functional genomics,
proteomics, bioinformatics (2000 - )

Nowadays Industrial biotechnology

... boosting as sustainable chemistry

Why choose for

industrial biotechnology?
Sometimes only way to make some products
Selectivity
Less by-products
Moderate reactor conditions
One process versus multiple chemical steps in chemical
synthesis (process intensification)
Sustainable: reduced energy, reduced CO2 emission (use of
renewable resources), reduced waste (biodegradable),
Economics (on a long term): fossil-based products become
more expensive due to increased prices of crude oil; in IB
biomass (agriculture and forest residues, energy crops, and
algae) can be applied as raw materials

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Why choose for

Industrial biotechnology?
Only way to (easily) make product

food fermentations
protein production
chemical synthesis is impossible or cumbersome (e.g., antibiotics)

Most selective process, less by-products

catalysis using enzymes from micro-organisms (e.g., aspartame)

Sustainability of the process

biofuels
biopolymers
bulk chemicals
enzyme-catalyzed processes

Industrial applications of
micro-organisms
Bioethanol
(yeast)

Enzyme production
(bacteria, moulds)

Production of biopharmaceuticals ,
antibiotics, vaccines (cell cultures,
recombinant micro-organisms)

Brewery fermentation tanks

Biological wastewater treatment

(activated sludge)

Markets
Fuel

Energy

Food & Beverages

Cosmetics and toiletries

Animal Feed

Alcohols

Pharmaceutical and

Plastics and fibers

nutritional products
Detergents and cleaning
products
Paper and pulp
Textiles

Biopharmaceuticals
Research & Biotechnology
(Environmental Biotechnology)

Product categories
Biochemicals
(e.g., food ingredients, biopharmaceuticals, bulk and fine
chemicals)

Biomaterials
(e.g., biopolymers)

Biofuels
(e.g., bioethanol, biogas (methane))

Enzymes

16

Examples of product from microbial conversion

Biopolymers

Biofuels
Biochemicals

Examples of product from microbial conversion

Products

Typical micro-organism(s)

Approximate worldwide
production [kg/year]

Bulk organic products

Ethanol

Saccharomyces cerevisiae

2. 1010

Acetone / butanol

Clostridium acetobutylicum

2. 106 (butanol)

Starter cultures

Lactic acid bacteria, bakers yeast

5. 108

Single-cell protein

e.g., Candida utilis

0.5-1. 108

Aspergillus niger

2-3.108

Corynebacterium glutamicum

3.108

Penicilline

Penicillium chrysogenum

3-4.107

cephalosporines

Cephalosporium acremonium

1.107

tetracyclines

Streptomyces aureofaciens

1.107

Biomass

Organic acids
Citrate
Lactate
Amino acids
L-glutamate
Antibiotics

Note: The following overview is a non-exhaustive list of products. Source: Doran, 1995

Examples of product from microbial conversion (cont.)

Products

Typical micro-organism(s)

Approximate worldwide
production [kg/year]

Extracellular polysaccharides
Xanthomonas campestris

5.106

Proteases

Bacillus spp.

6.105

-amylase

Bacillus amyloliquefaciens

6.105

Propionibacterium shermanii of
Pseudomonas denitrificans

1.104

Xanthan gum
Enzymes

Vitamins
B12
Vaccines
Tetanus

Clostridium tetani

Hepatitis B

Recombinant yeast cells

Therapeutic proteins
Insulin

Recombinant Escherichia coli

Examples of enzymes and their applications

Detergent enzymes
e.g., proteases, lipases,
carbohydrates

Food enzymes
e.g., glucose isomerase
[glucose into fructose
(sweetener)], pectinases
[fruit juice and wine
clarification, proteases
[baking - bread improver]

Textiles
e.g., catalase [bleaching],
proteases [desizing]
Source: Waites al. 2001

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FERMENTATION PROCESS

General layout plant

upstream processing, fermentation, downstream processing

24

Upstream processing:
strain selection/development
Bacteria and fungi (yeasts and moulds)
Escherichia coli
Bakkersgist
Bakkersgist

Aspergillus niger

Bacillus subtilis

Penicillium
chrysogenum

Micro-organisms as cell factories

Upstream processing:
strain selection/development
Product formation
Natural property (organic acids, alcohols, enzymes, antibiotics, vitamins,
amino acids, )

Enhanced natural property: dedicated modifications

to reactions of the cell metabolism to
increase/introduce desired product formation
(metabolic engineering)
Recombinant property: introduction of foreign DNA
(via recombinant DNA technology) to create
genetically modified strain secreting the desired
product (enzymes, therapeutic proteins, hormones, )
26

Upstream processing:
strain selection/development
Constraints

Nutritional requirements
Metabolic control
Shear sensitivity
Morphology
O2 requirements
Genetic stability
Metabolic by-products
Temperature optima
Viscosity

Note: Currently, tools for targeted

genetic engineering (metabolic
engineering) available for efficient
design of excellent GMOs.
However, GMOs are not generally
accepted (not in Europe) whereas
strains obtained via random
mutation techniques are.

High growth rate and production rate (yield and productivity!)

Easy processing

Upstream processing:
fermentation medium/raw materials
Source of carbon, nitrogen, phosphorous and sulphur, minor
elements, trace elements, growth factors, water, etc.
Design factors: metabolism of micro-organism, cost, availability,
stability, pretreatment requirement, presence of
contaminants/inhibitory components, sterilization,
Complex media versus minimal media

Mainly, use of high energy crops

Nowadays, shift towards use of agricultural and forestry residues

Upstream processing:
fermentation medium/raw materials
High energy crops, e.g., corn, maize,
cereals, sugar cane, beets

starch, glucose
Pretreatment, e.g. maize
milling
starch

gelatinization and saccharification (e.g.,
enzymatic hydrolysis, acid hydrolysis)
glucose.
Waste product from sugar industry
molasses.

Low energy plant waste, e.g., wood chips,

straw, waste paper, bagasse

lignocellulosic material (10-35 w/w %
lignin, 15-55 w/w % cellulose, 25-58 w/w %
hemicellulose)
Pretreatment, e.g., acid hydrolysis, enzymatic
hydrolysis.

Fermentation process systems

Suspended mode (cells dispersed in liquid) most frequent
Stirred tank reactor (e.g., enzyme production with Bacillus subtilis
(single cells), penicilline with Penicillium chrysogenum (flocs))

Cylindrical vessels (e.g., beer fermentation)

Bubble column
Airlift reactor
Supported mode (cells on carrier as biofilm)
Fluidized bed reactor (e.g., monoclonal antibody production with
animal cells)

Solid-state fermentation (cells on solid substrate)

Fermentation process systems

Selection and design considerations:
Often critical design issue: O2 supply
- efficient aeration and agitation
~ oxygen transfer rate [kg O2 /m.h]
- important energy cost
Shear sensitivity of cells
Efficient cleaning (CIP) and sterilization (SIP)
Control: temperature, pH
Foam production
Mode of operation

Note: sterilization with steam at 1 bar overpressure, at 121C, for 15-20 minutes

Scales of bioreactor systems

Different phases:
1.
Shake flask (mL)
2.
Lab-scale reactor (L)
3.
Pilot-scale reactor (0,3-10m3)
4.
Large-scale reactor (2-3000m3)

Stirred tank reactor

Typical configuration:
H/D from 1 to 3
Stirred
Aerated

Stirred tank reactor

Basic features
Reactor vessel (glass or stainless steel, sterile!)
On-line monitoring and control*:
Basic: temperature*, pH* (addition acid/base), agitation*, aeration*,
dissolved oxygen(*), off-gas, feed*
Advanced: biomass, substrate, metabolites
Heat exchangers:
cooling in large scale systems, heating in small-scale systems
internal for large-scale systems, external for small-scale systems
Mixing:
improve oxygen transfer and ensure homogeneity
stirrer design (e.g., Rushton turbines in bacterial fermentations)
baffles (enhance mixing, turbulence)
rotor entry on top (minimize risk of contamination)

Stirred tank reactor

Basic features (ctd)
Aeration
compressed air (or O2 enriched air)
sparger at bottom
aseptic (filter sterile)
Pressure
to increase dissolved oxygen concentration
to avoid contamination
Condenser on gas outlet (avoid medium loss due to evaporation)
Pumps (e.g., addition acid/base, substrate feed)
peristaltic pumps are recommended (aseptic pumping)
centrifugal pumps to be avoided (risk for contamination, high shear)
Foam removal
antifoam addition or mechanical foam breaker

Stirred tank reactor (5L)

BioFlo 3000
New Brunswick Scientific Inc.
@ Chemical and Biochemical Process
Technology and Control Section

Modes of operation
Industrial production processes
Batch
 Large flexibility; economical; low control
 Discontinuous operation (e.g., sterilization, filling,
cleaning)
Fed-batch
 Controlled growth/product formation rate (e.g., less
by-products, high cell concentration)
 Discontinuous operation
Continuous
 Highest productivity
 Sensitive for contaminations and genetic alterations in
production strain; process control

Airlift reactors
Basic features:
H/D ~ 6 - 10
No moving parts
Mixing due to expansion
of compressed air
Low shear and low
energy requirement

Solid-state fermentors

Downstream processing
Removal of cells
Centrifugation
Filtration

Recovery of product

Distillation
Chromatography
Dialysis
Centrifugation

Costs contributing to total production cost

High value,
value,
therapeutic
(recombinant)
products

Biomass,
Biomass, catabolic
products (ethanol,
citric acid, lactic
acid) [ = high
yield,
yield, low value
products]
products]

E.g., amino acids

vitamins,
vitamins,
antibiotics,
antibiotics,
intracellular
proteins [ = high
value,
value, low yield
products]
products]

Strategies for bioreactor process design as function

of cost-determining factor

SUSTAINABILITY OF IB-BASED PROCESSES

EXAMPLES: BIOPOLYMERS ENZYMES BULK
CHEMICALS - BIOETHANOL

Biopolymers
Examples
PTT (polytrimethyleneteraphtalate) on market
PLA (polylactic acid) on market
Bioisoprene in development
Motivation:
biodegradability
GHG emission decreases
Use renewable resources
Reduce energy inputs
Minimize pollution
Current limitations:
Development time
Cost
Properties

Biopolymers, e.g., PLA

Market dominated by 1 large player (Natureworks (Cargill))
Other companies involved are, e.g., PURAC (Holland), Hycail
(Finland), Galactic (Belgium, pilot with Total in Fluy of 1500
T/y)
Price ~ 2 Euro/kg (e.g. PE ~ 1Euro/kg)
PLA struggles with various problems property wise:
low glass transition temperature (55C)
high brittleness
poor barrier properties
poor thermal stability

Biopolymers, e.g., PLA

Ingeo (PLA) from Natureworks LLC

Manufacturing Ingeo produces 60% less

greenhouse gases than traditional polymers
like PET & PS

Manufacturing Ingeo uses 50% less nonrenewable energy (NREU) than traditional
polymers like PET & PS
http://www.natureworksllc.com

Enzymatic versus chemical catalysis

Cold enzymes in wash powders

Washing machines: big consumers
of household electricity, 80%
used to heat the water.
New generation of cold water
enzymes, T can be reduced from
40C to 30C, without sacrificing
cleanliness, and saving 30% of
the electricity used on the
laundry.
Also significantly reduced CO2
emissions: emissions can be
reduced by 100 g per wash by
washing at 30C rather than
40C2.

Bulk chemicals production

GHG savings per ton of IB chemical compared with their petrochemical
counterparts for current and future technology cradle-to-grave.

Herman et al. 2007

Bioethanol production

 Stages: (i) pretreatment of raw material (milling, hydrolysis), (ii)

fermentation (batch/continuous, aerobic/anaerobic, yeast/bacteria), and (iii)
separation (cell removal, distillation, drying)
Additive in gasoline (up to 10-20 v/v % ) < obliged by regulation
 Well-established production in US and Brazil (1st generation bioethanol)

Bioethanol production
First generation bioethanol: made from sugar, starch
Second generation bioethanol: made from
lignocellulosic material
 Pros of 2nd generation
GHG emission reduction
Reduced pressure on food crops
Reduced pressure on land use

 Current market place

fermentation

CO2 footprint

Advanced biofuels , Factsheet, EuropaBio 2009

Market penetration

Bioethanol

Bioethanol
To achieve industry expansion and cost reduction
- Improved pretreatment strategies: efficient hydrolytic
enzymes
- Stable and robust genetically-engineered micro-organisms:
able to convert C6 and C5, resistant to contaminants/inhibitors
in raw material, high productivity
- Efficient recovery of ethanol
- Valorization of side-products, e.g., recovery of lignin from
fermentation medium (as UV and sunlight additive for paints
and resins)
- Parallel businesses (integrated biorefineries) , e.g., Archer
Daniel Midlands (US) manufactures amino acids and food
ingredients besides bioethanol from corn, Dupont produces
1,3-PDO (propanediol) for biopolymers and residual
fermentation medium is used for bio-ethanol production.
e.g., Rogers et al. 2005.

References
Books on Industrial biotechnology & Bioreactor process engineering
P.M. Doran 1995. Bioprocess Engineering Principles. Academic Press Ltd, London (ISBN
0112-220856-0), 439 p.
M.J. Waites, N.L. Morgan, J.S. Rockey en G. Higton 2001. Industrial Microbiology: An
introduction. Blackwell Science, Oxford (ISBN 0-632-05307-0), 288 p.
References on sustainability of industrial biotechnology
Rogers et al. 2005. Application of biotechnology to industrial sustainability. Process Safety
and Environmental Protection, 83(B6):499-503.
Schmid et al. 2002. The use of enzymes in the chemical industry in Europe. Current
Opinion in Biotechnology, 13:359-366
EuropaBio Policy Guide 2010, Building a Bio-based Economy for Europe in 2020.
Nielsen P.H. 2005. Life Cycle Assessment Supports Cold-Wash Enzymes. International
Journal for Applied Science.
McKinsey, November 2010. Opportunities and challenges to develop the bio-chemical
industry. Presentation at Forum for Industrial Biotechnology (Flanders), CINBIOS, Mechelen
(Belgium).
Herman, Blok and Patel 2007. Producing Bio-Based Bulk Chemicals Using Industrial
Biotechnology Saves Energy and Combats Climate Change. Environmental Science and
Technology, 41:7915-7921
Advanced biofuels Factsheet, EuropaBio 2009