Nw biotech fundamentals day 2 session 2 industrial applications and cleantech

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Biotech Boot Camp
Session 2 – Industrial Applications of Biotechnology and
Cleantech
Presenter: Viktoriia Hristova
Melbourne, Brisbane, Sydney
28 May – 18 June, 2014

This session provides an overview of the biotechnology in
industrial biotechnology and cleantech
Analysis includes key biotech techniques and processes
and explains the various processes of one type of clean
energy, biofuels
The session also provides specific case studies and
examples on the evolution of cleantech and the industrial
applications of biotechnology
2
Session 2 Overview
Agenda

• Basic overview of industrial applications andcleantech
• The state of the art
• Regulatory nuances
• Future trends
• Case studies and examples
3
Session 2 Overview
Industrial applications and cleantech

• Basic overview of industrial applications and cleantech
• Future trends
4
Session 2 Overview

Industrial Biotechnology: The application of molecular biology
technology to modify the biological function of an organism to generate
industrial products and processes
• biomass-based materials such as fuels and chemicals, and
• the treatment of waste water and producing energy using more
efficient measures
Cleantech: includes recycling, renewable energy (wind power, solar
power, biomass and hydropower, biofuels), information technology,
green transportation, electric motors, green chemistry, lighting, and many
other appliances that are now more energy efficient. It is a means to
create electricity and fuels with a smaller environmental footprint and
minimise pollution
Industrial Applications and Cleantech
5
Basic overview of industrial applications and cleantech

• So....why the need for these areas of Biotechnology?
• Shift from petroleum-based economy
• Exhaustion and soaring price of petroleum
• Environmental issue
• Global warming (greenhouse gas, CO2 , emission)
• Pollution
• Development of renewable source-based bioprocess
• Replacement of chemical processes with bio-based ones
6

• Regulations are seen as restrictive to startups
• Cleantech is a child of innovation created by the
introduction of strict environmental regulations
7
Paradox

8
What is an eligible renewable energy source?
• hydro, wave, tide and ocean
• wind
• solar
• geothermal-aquifer
• energy crops
• wood waste
• agricultural waste and waste from processing of agricultural
products
• food waste and food processing waste
• Bagasse
• biomass-based components of municipal solid waste and landfill
gas
• sewage gas and biomass-based components of sewage

9
What is NOT an eligible renewable energy source?
• fossil fuels
• materials or waste products derived from fossil fuels

10
Fuel means any of the following:
(a) petrol
(b) automotive diesel
(c) liquefied petroleum gas
(d) liquefied natural gas
(e) compressed natural gas
(g) biodiesel (that is, a diesel fuel obtained by
esterification of oil derived from plants or animals)
(h) ethanol
(i ) any substance that is used as a substitute for a fuel
mentioned in paragraphs (a) to (h)
(j ) any substance that is supplied or represented as
(i) a fuel mentioned in paragraphs (a) to (h) or
(ii) a substitute substance under paragraph (i)

• Future trends
11
Session 2 Overview

• Design and construction of new biological parts, devices and systems
that do not exist in nature
• Also includes the re-design of existing, natural biological systems for
basic research and useful purposes
• Techniques widely used and adapted from Biotech for use in SB
• DNA synthesis tools
• De novo protein synthesis tool
• Multiplex Automated Genome Engineering (MAGE) and
• Omics
• It is the building block of GMOs which are then used to produce
biofuels
12
State of the art
Synthetic biology (SB)

• DNA synthesis tools
• Optimised synthetic sequences
• De novo protein synthesis tool
• Rational design
• Directed evolution
• Multiplex Automated Genome Engineering (MAGE)
• Enables rapid and continuous generation of sequence diversity at
many targeted chromosomal locations across a large population of
cells
• Omics
• Profiling techniques such as microarray and mass spec
13
State of the art
Synthetic biology (SB)

• Biomass
• Sustainable biomass = conversion of waste biomass to
energy
• Agricultural waste
• Bagasse from sugarcane
• Waste wood from timber mills
• House hold waste
• Algae
• Sewerage plants
14
State of the art
Biofuels

• Biomass technologies can be converted into energy through a range
of technologies. In Direct combustion, the most commonly used in
Australia, biomass is burnt to heat water, which in turn generates
steam for running a conventional generator
• Many biofuels are made from food crops, or other crops grown on
land that could be used to grow food. The world currently produces
more than 80 billion litres of fuel ethanol from these food crops
• Understandably, there has been a need to generate biofuels using
non-food woody crops, agricultural residues or harvest waste as
feedstock
15
State of the art
Biofuels

16
State of the art
Biofuels
Use of microorganisms (for biofuels or chemical
manufacture)
• Identify bacteria strain with high affinity for
decomposing ligno-cellulosic biomass
• Identify traits in other bacteria strains and
isolate DNA sequence
• Modify original bacterial DNA with desired traits
to enable its use in industrial processes of
Biofuel production at economic quantities

17
State of the art
Biofuel production

• Disadvantages
• finding an algal strain with a high lipid content and fast
growth rate that is not too difficult to harvest
• a cost-effective cultivation system that is best suited to that
strain
18
State of the Art
Biofuels- use of algae for biofuel production

19
State of the art
Biofuels- open pond use of algae for biofuel production

20
State of the art
Biofuels- open pond use of algae for biofuel production

21
State of the art
A breakthrough in 2013 was the
development of a rapid test for Lysteria
monocytogenes, a bacteria that causes food
poisoning, incorporates screen-printed
carbon electrode (SPCE) strips similar to
those used by diabetic patients for blood
glucose monitoring to measure glucose to
measure contamination in food
A gold-nanoparticle electrode sends the
signal to a hand-held device for presentation
similar to that for a glucose blood sample
Gold Nanoparticle-Modified Carbon Electrode Biosensor for the Detection of Listeria monocytogenes , Danielle
Davis, Xiao Guo, Leila Musavi, Chih-Sheng Lin, Sz-Hau Chen, and Vivian C.H. Wu., Industrial Biotechnology.
February 2013, 9(1): 31-36. doi:10.1089/ind.2012.0033.
Gold nanoparticle listeria biosensor

• Future trends
22
Session 2 Overview

23
Regulatory framework and formal guidance
Gene Technology Act 2000
Gene Technology Regulations 2001
GMO algae for fuel production

Gene Technology Act- object- Section 3
“The object of this Act is to protect the health and safety of
people, and to protect the environment, by identifying risks
posed by or as a result of gene technology, and by
managing those risks through regulating certain dealings
with GMOs”
24
Regulatory frame work and formal guidance

Regulatory Framework- Section 4
The object of this Act is to be achieved through a regulatory framework
which
(aa) Provides that where there are threats of serious or irreversible
environmental damage, a lack of full scientific certainty should not
be used as a reason for postponing cost effective measures to
prevent environmental degradation and
(a) Provides an efficient and effective system for the application of
gene technologies; and
(b) Operates in conjunction with other Commonwealth and State
regulatory schemes relevant to GMOs and GM products
25

The National Industrial Chemicals Notification and
Assessment Scheme (NICNAS) assesses new and existing
industrial chemicals, including genetically modified products,
for their effects on human health and the environment
26
Regulatory bodies- NICNAS

• What are dealings?
• ‘Deal with’, in relation to a GMO, means the following
• conduct experiments with the GMO
• make, develop, produce or manufacture the GMO
• breed the GMO
• propagate the GMO
• use the GMO in the course of manufacture of a thing that is not the
GMO
• grow, raise or culture the GMO
• import the GMO
• transport the GMO
• dispose of the GMO
• and includes the possession, supply or use of the GMO for the
purposes of, or in the course of, a dealing mentioned in any of those
listed above
Industrial Applications and Cleantech
27
Regulatory nuances

GMO register
Dealings with GMOs may be entered on the GMO Register
once they have been licensed for a certain period of time.
Dealings will not be entered onto the Register until the
Regulator is satisfied that the dealings are safe enough that
they can be undertaken by anyone, and that safety does not
depend on oversight by a licence holder.
28
Regulatory process- definitions

Licences
All dealings with GMOs (that are not exempt, Notifiable Low Risk
Dealings (NLRD) or already on the GMO Register) need to be licensed
by the Regulator
The licensing system is based on rigorous scientific risk assessment and
extensive consultation with expert advisory committees and government
agencies—and for intentional releases of GMOs into the environment,
the public
The GTR has 90 working days in which to make a decision to either
issue, or refuse to issue, a licence for the dealings proposed in a DNIR
application
29

Exempt dealings
Certain types of dealings with GMOs involve a very low risk
(i.e. contained research involving very well understood
organisms and processes for creating and studying GMOs).
Other than listing in the Regulations, the only legislative
requirement for exempt dealings is that they must not involve
an intentional release of a GMO into the environment
30

Notifiable Low Risk Dealings- NLRD
The Regulations also set out categories of dealings with
GMOs which are low risk and may proceed if certain
conditions are observed, such as specified dealings only
being undertaken in certified contained facilities, overseen
by Institutional Biosafety Committees and notified annually to
the Regulator
An NLRD must not involve the intentional release of a GMO
into the environment
31

Emergency Dealing Determination
The Minister may make an EDD authorising dealings with
GMOs for a limited time in an emergency
The Minister must be satisfied that
(a) there is an actual or imminent threat to people or the
environment
(b) that the EDD would adequately address the threat and
(c) that the risks posed are able to be managed to protect
people and the environment
The Minister must receive advice from the Regulator about
managing these risks
32

• Industrial use of a modified microorganism for fuel or
chemical production would require a license
• Use of modified algae would require a license
• Use of modified algae in open-pond or similar outdoor
reactor would require a DIR (Dealing Involving Release)
license from the GTR
• Procedures for review of DIR license applications are
similar to the DNIR license (longer assessment phase of
255 working days)
33
Regulatory nuances
Examples - GMO algae for fuel production

• Applications lodged
• Reviewed by Institutional Biosafety Committee (IBC)
• Submitted to Gene Technology Register (GTR)
• GTR prepares Risk And Risk Management Plan
(RARMP)
• Public consultation on RARMP
• Decision on licence
• Post-approval procedures
34
Regulatory nuances
GTR licensing process

Fuel Quality Standards Act 2000
Fuel Quality Standards Regulations 2001
Fuel Standard (Biodiesel) Determination 2003
Fuel Standard (Ethanol E85) Determination 2012
Renewable Energy (Electricity) Act 2000
Renewable Energy (Electricity) (Large-scale Generation
Shortfall Charge) Act 2000
Renewable Energy (Electricity) (Small-scale Technology
Shortfall Charge) Act 2010
Renewable Energy (Electricity) Regulations 2001
35
Regulatory nuances
Fuels and renewable energy regulations

The Fuel Quality Standards Act – object- Section 3
(a) regulate the quality of fuel supplied in Australia in order to
(i) reduce the level of pollutants and emissions arising from the
use of fuel that may cause environmental and health problems
and
(ii) facilitate the adoption of better engine technology and
emission control technology and
(iii) allow the more effective operation of engines and
(b) ensure that, where appropriate, information about fuel is
provided when the fuel is supplied
36

Regulatory Framework- objective
The standards regulate the quality of the fuel supplied to
consumers and reduce toxic vehicle emissions. Modern
vehicles require fuels that meet high quality standards and
can be damaged by inferior products. The production of
cleaner fuels will result in cleaner air which will have an
impact on society’s health, the local and global
environments, and reduce the economic and social costs of
illnesses linked to vehicle pollution
37
Regulatory nuances

Fuel Standards Consultative Committee
• The Fuel Standards Consultative Committee (the Committee) was
established under the Act as a formal consultation mechanism
• The Minister must have regard to the Committee's recommendations
about a range of matters. The Minister must consult the Committee
before:
• determining a fuel standard or fuel quality information standard
• granting, varying or revoking an approval to vary a fuel standard for
a specified period
• entering or removing contents from the Register of Prohibited Fuel
Additives and
• preparing guidelines for more stringent fuel standards which may
apply in specified areas in Australia
38
Regulatory nuances

• Commonwealth inspectors in all states and territories
conduct sampling along the supply chain
• Samples tested at National Association of Testing
Authorities (NATA)- independent lab
• Industry Notification and reporting
39
Regulatory nuances
Monitoring and compliance process

• All fuel suppliers (importers, producers and distributors)
required to provide the following documents relating to
the fuel being supplied
• Statement on whether the fuel meets the relevant fuel
quality standard
• Information about the supplier
• Information about the product
• The delivery docket number (if relevant)
• Industry’s documentation and related production and
distribution processes are likely to be investigated if non-
compliant fuel is detected
40
Regulatory nuances
Industry notification and reporting

Regulatory Framework
The petrol and diesel standards were an essential step in implementing
the Commonwealth Government's commitment to facilitate the adoption
of better, cleaner emission control technology, the more effective
operation of engines, and to reduce pollution and vehicle emissions. That
work has continued with the development of the Fuel Standard (Autogas)
Determination 2003, Fuel Standard (Biodiesel) Determination 2003, Fuel
Quality Information Standard (Ethanol) Determination 2003, Fuel
Standard (Ethanol E85) Determination 2012, and the Fuel Quality
Information Standard (Ethanol E85) Determination 2012
41
Regulatory nuances

Fuel Standard (Biodiesel) Determination 2003
• The Fuel Standard (Biodiesel) Determination 2003
defines biodiesel as ‘a diesel fuel obtained by
esterification of oil derived from plants or animals’. Put
simply, it is a fuel derived from plant and/or animal matter
rather than petroleum sources
• Internationally, biodiesel is used as a transport fuel in its
own right (known as B100) or in blends with petroleum
diesel. Most common blends used are 5% biodiesel (B5)
and 20% biodiesel (B20)
42

The new E85 standards are:
• Fuel Standard (Ethanol E85) Determination 2012 – this
standard specifies the physical and chemical parameters
for E85, and testing methods to determine compliance
with the standard
• Fuel Quality Information Standard (Ethanol E85)
Determination 2012 – this standard sets out the labelling
requirements for the sale of E85 fuel sold in Australia
43

Monitoring
Authorised fuel inspectors undertake fuel sampling
nationally, at all stages of the fuel supply chain. Any fuel
subject to a fuel standard under the Fuel Quality Standards
Act will be tested, including E85. Samples are regularly
taken from importers, refineries, distributors and service
stations. Inspectors check for E85 labelling during their
inspection.
44

Renewable Energy (Electricity) Act 2000 – object- Section 3
(a) to encourage the additional generation of electricity from
renewable sources and
(b) to reduce emissions of greenhouse gases in the
electricity sector and
(c) to ensure that renewable energy sources are ecologically
sustainable
The objective of this Act is achieved through the issuing of
certificates for the generation of electricity using eligible
renewable energy sources
45

• Future trends
46
Session 2 Overview

• Development of high performance cell lines for the
biological manufacture of fuel ethanol, industrial enzymes
and pharmaceutical products
• Conversion of industrial waste gases into fuels
• Modified yeast strain that increases ethanol production
• Biosynthesis of chemicals and fuels from renewable
resources such as CO2
47
Future trends

• Future trends
48
Session 2 Overview

49
Case Studies and Examples
BioMine*
Development of bacteria capable of surviving within high
salinity/ high stressor environments
The unmet need
Fuel ethanol is produced by the fermentation of a
carbohydrate substrate by yeast cells. In this process,
yeast is severely inhibited in reaching its full productive
potential by a variety of stresses including rising alcohol
concentration, low pH, high temperature, bacterial
contamination, and dissolved chemicals
* Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 2013
R&D Tax Incentive Biotechnology Guidance Example in context

50
Real-life example- iDiverse
iDiverse is a biotechnology company focused on providing
gene technology for developing high-performance cell
lines used in the bioproduction of fuel ethanol, industrial
enzymes, and pharmaceutical products; and for creating
transgenic plants that are resistant to a broad spectrum of
diseases and environmental stresses
Companies developing modified microorganisms for
ethanol production

51
The Technology
The technology includes genetic sequences which, when
placed in cells, allow the resulting transformed cells to fend
off a variety of stresses that occur in the bioproduction
process.
This enables the cells to produce more product (ethanol,
enzyme, or pharmaceutical) than comparable cells lacking
this genetic sequence
ethanol production

52
The Technology
As a prelude to testing the sequences in the production of
fuel ethanol, iDiverse subjected yeast transformed with
several of their sequences to the surrogate stress of
hydrogen peroxide (H2O2), which is highly toxic to living
cells
In the following slides is the results of the experiment that
show yeast transformed with the iDi-032 sequence tested
against control yeast not containing the sequence
ethanol production

53 http://www.idiverse.com/html/tech_bioproduction.htm
The Results Yeast cells not stressed with Hydrogen
peroxide
ethanol production

The Results Yeast cells stressed with 5mM Hydrogen
peroxide
ethanol production

The Results
1st graph- Yeast containing the iDi-032 gene grows at the
same rate and produces relatively the same number of
cells over a 48-hour time course as does the control yeast
when there is no peroxide stress applied
2nd graph- under the influence of 5mM H2O2, growth of
both cell cultures was suppressed during the first 24 hours
and then grew to different levels in the next 24 hours with
the iDi-032 transformed cells growing to a cell count
roughly 3x higher than the controls
ethanol production

The benefits
• The technology is applicable to current fuel ethanol manufacturing
processes using corn and sugar cane as starting materials and also to
those being developed to use cellulosic biomass
• Use of iDiverse cell lines incorporating their proprietary ProTectAll™
transgenic technology have enhanced resistance to the stresses of the
bioproduction process
• This will enable these cells to produce more product at higher
concentrations, using less nutrients, and under more extreme
conditions. All contributing to higher production efficiencies at lower
costs
56
Case studies and examples
ethanol production

• LanzaTech
• NZ company, and a leader in gas fermentation technology.
• It provides novel and economic routes to fuels and high
value chemicals from industrial wastes and residues such
as industrial flue gases from steel mills and other
processing plants; syngas generated from any biomass
resource such as MSW, organic industrial waste and
agricultural waste and reformed methane residues
57
Companies developing modified microorganisms for ethanol
production & conversion of industrial waste gases into fuels

The unmet need
• Need to produce Biofuels from non-food crops
• LanzaTech’s technology solutions mitigate carbon emissions from
industry without adversely impacting food security or causing indirect
land use change
• The technology reduces overall GHG emissions as it substitutes
carbon from fossil fuels. This is a much more efficient use of carbon,
then turning it into power and reduces emissions of harmful air
pollutants such as NOx and particulates up to 90%
58

The Technology
• Gas feedstock is scrubbed, cooled and sent to a bio-
reactor
• Carbon component is used as a food source for
LanzaTech microbes
• These microbes undergo biofermentation which
produces ethanol as a liquid biofuel
59

The Technology
60 www.lanzatech.com

The Technology
• LanzaTech’s gas fermentation process uses carbon-
containing gases as both a nutrient and energy source
for its proprietary microbes to produce fuels and
chemicals
• The platform is able to utilize gases that contain carbon
monoxide (CO), with or without hydrogen (H2), or gases
containing carbon dioxide (CO2) and H2, providing a
novel approach to carbon capture and reuse
61

The Benefits
• In 2012, LanzaTech reached key development
milestones when it became the first company ever to
scale gas fermentation technology to a pre-commercial
level.
• Working closely with its partner in China, Baosteel,
LanzaTech developed and successfully operated a
facility with an annualized capacity of 100,000 gallons of
ethanol and is currently operating a second facility (also
100,000 gal/year), with Capital Steel in Beijing
62

63
TimbaFuels*
Development of technology in improving the extraction
yield of ethanol from lingocellulose feedstock for
generation of biofuels
• Another example is Xylogenics Inc – developed a
modified yeast strain which can use corn kernels, corn
stover, wheat straw, barley straw, grasses, wood
waste and public waste to produce biofuel
* Fictitious example from AusIndustry R&D Tax Incentive Biotechnology Guidance product, April 2013
R&D Tax Incentive Biotechnology Guidance Example in context

• Xylogenics Inc
• Spin-out from Indiana University Medical School
• Developed a bioengineered yeast that breaks down
cellulose to produce ethanol
• The bioengineered yeast can be genetically optimized for
specific sources such as corn or woodchips
• The company says that this yeast strain is able to
increase ethanol production from cellulose by at least
30%, while also allowing producers to use feedstock such
as corn kernels, corn stover, wheat straw, barley straw,
grasses, wood waste and public waste
64
Modified yeast strain increases ethanol production by 30%

• EASEL Biotechnologies LLC
• Cutting-edge, sustainable platform technologies through
the science of metabolic engineering
• Genetically modified E.coli make higher alcohols (3-8
carbon atoms) from glucose or directly from carbon
dioxide
• Alcohols further processed to produce green fuels
65
Biosynthesis of chemicals and fuels from renewable
resources such as CO2

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