industrial crop platforms for the production of chemicals
TRANSCRIPT
INDUSTRIAL CROP PLATFORMS FOR THE PRODUCTION OF
CHEMICALS AND BIOPOLYMERS
Outputs from the EPOBIO project April 2007
Prepared by Jan B. van Beilen, Ralf Möller, Marcel Toonen, Elma Salentijn,
and David Clayton
Flagship leader: Yves Poirier, Bill Orts
Series editor: Dianna Bowles
cplpressScience Publishers
EPOBIO: Realising the Economic Potential of Sustainable Resources - Bioproducts
from Non-food Crops
© April 2007, CNAP, University of York
EPOBIO is supported by the European Commission under the Sixth RTD
Framework Programme Specific Support Action SSPE-CT-2005-022681 together
with the United States Department of Agriculture.
Legal notice: Neither the University of York nor the European Commission nor any
person acting on their behalf may be held responsible for the use to which
information contained in this publication may be put, nor for any errors that may
appear despite careful preparation and checking. The opinions expressed do not
necessarily reflect the views of the University of York, nor the European
Commission. Economics data for this report was provided by Cranfield University,
UK and the input of Dr. Anil Graves and Prof. Joe Morris is acknowledged. We also
thank Prof. Klaus Ammann for providing information on geneflow and outcrossing.
Non-commercial reproduction is authorized, provided the source is acknowledged.
Published by:
CPL Press, Tall Gables, The Sydings, Speen, Newbury, Berks RG14 1RZ, UK
Tel: +44 1635 292443 Fax: +44 1635 862131 Email: [email protected]
Website: www.cplbookshop.com
ISBN 10: 1-872691-23-4 ISBN 13: 978-1-872691-23-7
Printed in the UK by Antony Rowe Ltd, Chippenham
i
CONTENTS
EXECUTIVE SUMMARY V
1 INTRODUCTION 1
1.1 Platform chemicals and polymers from renewable materials 2 1.2 Production of platform chemicals and polymers in planta 2 1.4 Selecting crops for the production of platform chemicals and biopolymers 7
2 THE POLICY CONTEXT FOR THE BIOECONOMY 9
2.1 Introduction 9 2.2 Biorefineries and the bioeconomy 10 2.3 Fossil oil and biofuels 11 2.4 Common Agricultural Policy (CAP) 12 2.5 Use of genetically modified plants 14 2.6 Land use and availability 15 2.7 Climate change 15 2.8 Sustainable development 16 2.9 Developing countries 17 2.10 Industrial competitiveness 18 2.11 Strategic conclusions and recommendations 19 2.12 Specific conclusions and recommendations for the biopolymers and
platform chemicals crop platform 20
3 SUGAR BEET (BETA VULGARIS) 22
3.1 Introduction 22 3.2 Beet processing 24
3.2.1 Sugar beet processing 24 3.2.2 Conventional processing steps used for sugar crystallisation 25 3.2.3 Side products of conventional sugar beet processing 29 3.2.4 Biofuels and beet technical quality 31 3.2.5 Novel co-products and processing technology 33
ii
3.3 Genetics 35 3.3.1 Taxonomy 35 3.3.2 Genetics 37 3.3.3 Tools 37
3.4 Breeding 38 3.4.1 Historical overview and background 38 3.4.2 Conventional and marker assisted breeding 39
3.5 Susceptibility to abiotic stresses 40 3.6 Susceptibility to biotic stresses 41 3.7 Agronomy 41 3.8 Environmental impacts 43
3.8.1 Agronomical impacts and water requirement 43 3.8.2 CO2 emission and carbon sequestration 45 3.8.3 Gene flow and biosafety 47
3.9 Economics 51 3.9.1 Cultivation costs and net margins 51 3.9.2 Bioethanol 54 3.9.3 Production of chemicals and biopolymers in beet 56 3.9.4 Development and registration costs of GM-beet 56
3.10 SWOT analysis 57 3.11 Research and development needs 58
3.11.1 General R&D needs 58 3.11.2 Specific ideas from reports and scientific literature 59
4 TOBACCO (NICOTIANA TABACUM L.) 63
4.1 Introduction 63 4.2 Current and future co-products 66
4.2.1 Biopharmaceutical proteins and vaccines 66 4.2.2 Industrial enzymes 67 4.2.3 Polymers 68 4.2.4 Platform chemicals 69 4.2.5 Plant oils 71
iii
4.2.6 Processing of tobacco for biopolymer and platform chemical
production 72 4.3 Genetics 75 4.4 Breeding 76 4.5 Susceptibility to abiotic stresses 78 4.6 Susceptibility to biotic stresses 79 4.7 Agronomy 79 4.8 Environmental impacts 81
4.8.1 Agrochemical inputs, nutrient and water requirement 81 4.8.2 CO2 emission and carbon sequestration 85 4.8.3 Gene flow and biosafety 85
4.9 Economics 86 4.9.1 Yield 86
4.9.2 Competitiveness of plant-produced industrial products 87 4.10 SWOT analysis 88 4.11 Research and development needs 89 5.1 Introduction 91 5.2 Potential co-products 93
5.2.1 Introduction 93 5.2.2 Production of biopolymers 94 5.2.3 Production of platform chemicals 95
5.3 Miscanthus processing 97 5.3.1 Polymer extraction 97 5.3.2 Chemicals 98
5.4 Breeding and genetics 99 5.4.1 Introduction 99 5.4.2 Classical and marker assisted breeding 100 5.4.3 Genetic transformation 102
5.5 Susceptibility to abiotic stress 102 5.6 Susceptibility to biotic stress 103 5.7 Agronomy 103
5.7.1 Establishment 103
iv
5.7.2 Cultivation 105 5.7.3 Harvesting 105 5.7.4 Conversion to other crops 107 5.7.5 Biomass yields 107
5.8 Environmental impacts 109 5.8.1 Agrochemical inputs, nutrient and water requirement 109 5.8.2 CO2 emission and carbon sequestration 109 5.8.3 Gene flow and biosafety 111
5.9 Economics 111 5.10 SWOT analysis 115 5.11 Research and Development needs 116
EPOBIO RECOMMENDATIONS 118
REFERENCES 125
INSTITUTIONS, ORGANISATIONS AND PERSONS CONTACTED 151
v
EXECUTIVE SUMMARY
EPOBIO is an international project to realise the economic potential of plant-derived
raw materials by designing new generations of bio-based products that will reach
the marketplace 10-15 years from now.
EPOBIO is a "science-to-support-policy" project funded by the Framework 6
programme of the European Commission (EC). Partners from the European Union
(EU) and United States (US), from academic research institutions and from industry,
work together with an International Advisory Board of researchers, industrialists and
policymakers. The aim is to ensure a robust and holistic evidence-base is
established to inform future national and international decision-making. This
"EPOBIO process" considers new science-led projects and products within a wider
context of their environmental impact, economics, regulatory framework, social
acceptability and expectations of the public and policymakers. This holistic process
underpins strategic recommendations that constitute the major outputs of EPOBIO.
The ability of plants to capture solar energy and use carbon dioxide and water to
photosynthesise carbohydrates offers the potential of a sustainable manufacturing
system. Crop plants already provide cheap commodity chemicals such as starch
and sugar. The possibility of establishing industrial crop platforms for the
production of a wider range of commodity chemicals and polymers needs to be
explored, particularly at a time when alternatives to petrochemicals must be found.
The security and cost of supply of fossil reserves, together with the environmental
impacts of climate change, are driving the search for sustainable alternatives.
Substantial quantities of fossil reserves are specifically used as petrochemicals to
make a vast range of items from pharmaceuticals to plastics, agricultural fertilisers
and many different consumer products.
Due to these issues of security and cost of feedstocks, coupled with the urgency to
establish sustainable manufacture, the chemical industries globally are increasingly
vi
seeking alternatives to the use of petrochemicals. There are two alternatives based
on agricultural feedstocks in current practice. One involves chemical synthesis,
such as the use of the Fischer Tropsch process from bio-based source materials,
the production of levulinic acid from cellulose and polyols from sugars. The other
alternative uses microorganisms and microbial processes to produce industrial
chemicals from agricultural feedstocks by fermentation and biotransformations of
plant products such as starch, sugar, and plant oils or co-products and waste. This
is the well-established route of industrial biotechnology and forms the chosen
process for many products already on the market.
A third route is the use of crop plants to produce novel industrial chemicals in the
field, whether finished product for extraction or precursors for post-harvest
modification into product. This route of using industrial crops for large-scale
production of commodity chemicals and polymers is not yet in widespread use,
beyond the traditional examples of products produced naturally by plants such as
sugars, starches, natural rubber and the oils produced by oil crops.
This report addresses the third route and explores the feasibility of using crops for
the production of novel industrial chemicals and biopolymers. The report
complements "Natural Rubber", prepared by EPOBIO in 2006 and available at
www.epobio.net.
Three potential industrial crop platforms for commodity chemical production are
considered. These are crops already of relevance or of great promise to agriculture
in the Member States of the EU and case studies are developed to explore their
potential as new platforms for chemical manufacture. The crops are sugar beet, the
perennial grass and energy crop Miscanthus and tobacco. The principal question
addressed in this study is the feasibility of producing chemicals and biopolymers
more cheaply in fields than in bioreactors within the timespan of 10/15 - 20 years.
All of the applications described in this study for industrial-scale production of novel
chemicals in field crops necessitate the use of genetic modification (GM). It is up to
vii
public acceptance whether these new transgenic crops will be developed and
cultivated in Member States throughout the EU.
For each of the crop platforms, the current state-of-the-art is reviewed with a
detailed bibliography. The research and development (R&D) needs are identified in
terms of the work that will need to be undertaken to achieve an optimised platform.
Full consideration is given to recommendations for each of the crops and the
possibility that the platforms can be used for multiple products - such as biomass for
bio-energy as well as valuable co-products for extraction and processing in
biorefineries.
Sugar beet
A strength of sugar beet is that it is already an established crop throughout Europe,
is a high income generator for the farming community and has exceptional yields of
dry biomass per hectare. There is also a considerable science-base underpinning
the crop and its current use for production of refined sugar and co-products for the
food and feed markets.
Sugar beet, as a producer of sugar, or its close relative fodder beet, is already a
feedstock for bio-energy biorefineries, and this use would be further optimised if
biomass production and the yield of fermentable materials including sugars were
optimised. The R&D needs in the context of industrial use of beet are very different
from those that have underpinned development of the crop to date and will need to
be refocused urgently if the potential of beet as an energy and industrial crop were
to be pursued widely in the EU.
This study is considering sugar beet as an industrial platform for commodity
chemicals/biopolymers beyond its potential use as simply a bioenergy crop. It must
be emphasised that this extended use for chemical production would necessitate
development and field cultivation of transgenic varieties. These varieties would be
engineered as appropriate for the specific product(s) that the beets are designed to
viii
manufacture. However, in relation to the production of industrial transgenic beet, it
is an absolute essential to develop technologies to prevent transgene flow, given
the considerable risk from outcrossing and consequent transgene spread.
Also, processing of sugar beet for refined sugar and co-products has been
extensively optimised. New processing schemes are already under development for
use of sugar beet in the production of energy products such as bioethanol or
biobutanol. Should the beet be developed further for multiple uses that combine
bioenergy with production of novel chemicals it is highly probable that processing
technologies would have to be still further modified and newly designed. For low
value co-products, these changes should be minor and relatively easy to implement;
for high value-added products, processing would focus on the main novel product
with waste streams feeding into bioenergy or biofuel production.
Thus, whilst technologies are increasingly available for development of beet as an
industrial crop platform with multiple outputs, there are a number of weaknesses
that must be addressed. These range from inherent difficulties of developing a
transgenic crop used for both non-food and food purposes, the need to prevent
transgene flow, and the high inputs currently needed for high yield. The locked
supply chain in place for sugar refining can be considered both as an advantage
and a disadvantage since there are some processors already keen to look for
alternative uses.
With reform of the Sugar Regime within the Common Agricultural Policy (CAP) it is
probable that sugar refining from sugar beet will decline throughout the EU in the
next few years, but the crop may be maintained for use in biofuel development.
These changes will open up the opportunity to develop new markets for the crop.
Given the agronomic expertise available in many Member States for the cultivation
of beet, and the technologies available for crop improvement, increased
sustainability and novel modification, development of beet as a new industrial
platform should reasonably be examined, and indeed this process is already
underway.
ix
Tobacco
This is currently grown as a field crop in many Member States of the EU and in
those regions extensive agronomic experience exists. CAP support for the
production of tobacco is being switched from direct support to incorporation into the
single farm payment, opening up access to new markets. In addition, financial
allocation for restructuring in tobacco growing regions also supports the possibility
of alternative uses for tobacco as an industrial crop platform.
Alternative uses of tobacco are already in development in that the plant is used for
the production of biopharmaceutical proteins in leaves of transgenic tobacco grown
in containment. This study raises the possibility of widening the applications of
transgenic tobacco to field crop cultivation and its use as an industrial platform for
chemicals and biopolymers. In this context, tobacco benefits considerably from
established genetics and its use as a laboratory tool which has led to robust
protocols for genetic transformation, notably also of the chloroplast.
Research and development should focus on the nature of the chemicals and
polymers chosen for production in tobacco. Since tobacco contains little dry matter
and is currently unlikely to represent a biomass crop it is probable that bespoke
transgenic lines would be developed for each chemical and biopolymer product.
Given the costs associated with development of transgenics, it is likely that these
products would be mid- to high-value soluble chemicals and polymers, including
enzymes. Production of hybrids with other Nicotiana species could be a route to
increase biomass leading to both increased yields of novel chemical products and
increased residual biomass that could be used for fuel generation. In view of the
dilute nature of this waste stream, this would most likely be biogas.
Clearly, tobacco is already used as a non-food crop and has no related species in
Europe and North America. These features greatly limit the risks from outcrossing
and therefore, transgene flow to food crops is not an issue. As a crop grown on
relatively limited hectarage, transgenic tobacco would be relatively easier to isolate
x
than other large-scale biofactory crops. Indeed, since tobacco offers versatility in
terms of production, R&D should also be directed towards the design and
development of small-scale extraction and processing protocols, such that on-farm
post-harvest treatments could be both feasible and profitable for small-scale
producers and contribute to rural development in tobacco growing regions of the
EU.
Miscanthus
The perennial grass, Miscanthus, has substantial strengths in terms of yield
potential and ability to grow successfully under low inputs of fertiliser and pesticides.
Miscanthus is already recognised to present a considerable opportunity for
bioenergy production, given parameters such as biomass yield and low inputs.
However, its use for bioenergy is currently severely limited because the grass is not
developed as yet as a crop for widespread cultivation. In due course it is likely that
experience with related grasses such as sugar cane, maize and Sorghum will
greatly benefit the development of Miscanthus.
Research needs are those associated with any plant species that is undeveloped as
an agricultural crop. There are urgent needs to improve our understanding of the
genetics of Miscanthus, to establish a robust breeding programme and to develop
molecular tools for fast-track breeding. Research is also required to establish a
robust genetic transformation system for Miscanthus. In this context, parameters
for successful tissue culture systems need to be optimised for regeneration
purposes.
In terms of agronomy, Miscanthus is not completely frost-tolerant, with particular
issues in the first winter following establishment. Improvements to the crop are
required to increase frost hardiness, which in turn would greatly expand the
cultivation areas suitable for Miscanthus across Europe. Whilst the grass has
considerable yield potential, productivity under low input conditions is another target
for improvement. Current practice is the use of rhizomes to establish Miscanthus.
xi
This is labour-intensive and new approaches need to be optimised such as seed
sowing or field establishment at the plantlet stage.
In addition to these many R&D needs to establish the grass as a regular agricultural
crop, there would be the added needs to establish its potential as a platform for
chemical and polymer production to complement biomass use for bioenergy. This
added potential will depend on the development of a robust transformation system
and much greater understanding of metabolic pathways in the perennial grass to
design appropriate change in flux into the novel products, without impacting greatly
on biomass yield. There are also R&D needs in terms of extraction methods for
application in biomass biorefineries. However, the feasibility of using Miscanthus for
the production of chemicals or biopolymers can be judged from current
developments with sugar cane.
Strategic recommendations - science
Recommendations within this theme of industrial crop platforms for the production
of commodity chemicals and biopolymers, must be viewed from the perspective of
underpinning work that needs to be undertaken to ensure products in a market
place in a 10/15 � 20 year time period. Currently, the economics of field production
versus bioreactor production lead to commercial decisions to manufacture
chemicals and biopolymers such as polyhydroxyalkanoates (PHA), by fermentation
routes. The EPOBIO report has explored the potential for new field crops to produce
these chemicals and biopolymers and considers what would be needed to develop
three potential new industrial crop platforms for this purpose.
The key question that arises is whether it is appropriate to design any plant platform
to make novel commodity chemicals/polymers. Should the community rather focus
on platforms to manufacture cheaper and more efficient biofuels from biomass, as
well as the more simple feedstocks produced naturally by plants to support the
bioreactor production of industrial commodities?
xii
The report has considered three quite different crops: sugar beet, tobacco, and
Miscanthus. The strengths and weaknesses of developing each of these three
crops as future industrial crop platforms are quite different.
The study has revealed the potential of sugar beet to be optimised as an industrial
energy crop that could be further modified to produce platform chemicals. The
extensive area on which this crop is already cultivated in Europe and the opening of
markets through CAP reform are two positive issues that would underpin new
development of the crop into an industrial platform. Since sugar beet is currently
considered and used as a food crop linked into integrated supply chains for sugar
refining, a substantial change in perception would be needed before alternative
uses could be taken up. These changes in perception are already occurring, with
sugar beet increasingly in use as a biomass crop for bioenergy. Current industrial
applications of sugar beet are based on beet that has been optimised for sugar
refining. Industrial utility of the crop would be greatly enhanced if new breeding
targets aimed at industrial applications were undertaken. Beyond bioenergy, there
are opportunities for using beet to produce novel chemicals and biopolymers.
However, social acceptability of transgenic beet for this purpose is likely to play a
major determining role in decisions.
Tobacco offers interesting potential as an industrial crop and there is extensive
agronomic experience with the crop from farmers who already produce tobacco
within the EU. This is a transgenic application, but tobacco has many strengths for
high yield production of designer compounds by GM and the possibility for
development into a relatively high yielding biomass crop. Given the cost of the
development of a transgenic crop it is likely that the considerable potential of
tobacco as an industrial platform will be pursued primarily by the large
biotechnology companies. The extent of cultivation of GM across member states of
the EU will depend on public acceptability. In the longer term, there may be scope
for on-farm processing of relatively small hectarage, which could provide alternative
uses of the crop for existing producers.
xiii
Miscanthus undoubtedly holds great promise as a bioenergy crop for the mid- to
long-term future. This promise can only be realised once the grass has been
optimised for large-scale commercial cultivation. Miscanthus offers potential for co-
production of added value products in parallel to biomass for biofuels.
STRATEGIC RECOMMENDATIONS – POLICY
Our strategic recommendations on policy encompass six specific elements to
ensure take up of the bio-based economy. These are:
! Policies must be coherent, integrated and coordinated.
Integration in Brussels and Member States is essential to develop a policy
framework that will support the bioeconomy. As the bioeconomy represents a
potentially huge strategic development consideration should be given to applying a
�bioeconomy test� to policies in development, in the same way that policies are
assessed for their sustainable development impacts.
! Innovation in plant and industrial biotechnology should be supported.
Clear research objectives and a framework to achieve them are essential. An
adequate level of targeted funding, selecting those novel and innovative processes
and products likely to achieve success in the market place and deliver
environmental benefit, should be an element of this.
! Policies should support development of the whole supply chain.
This will need to consider feedstock supply, processing and the production of
bioproducts. There is a need to both stimulate the market side and build on the
foundation of the Common Agricultural Policy, which has moved from production
subsidy to market-orientated developments. Financing along the supply chain
needs to be considered as one aspect of feedstock supply.
xiv
! A communication strategy is essential.
The acute lack of awareness of the bioeconomy and the potential of biotechnology
at all levels in society must be addressed by a strategic communications campaign
designed to raise awareness and create an informed acceptance of bioproducts.
This will need to explain the benefits of the processes and products delivered by the
bioeconomy.
! Pilot projects have a role to play.
The establishment of proof of concept and testing under industrial conditions is a
key step in moving research into product development. Scale-up during the
research phase can develop and test industrial processes and also help to develop
stronger co-operation between industrialists and academics.
! Measurable sustainability indicators should be developed.
The absence of validated techniques for the measurement of sustainability benefits
needs to be addressed. This is important as these gains need be evidenced to
enable all stakeholders to understand the rationale for the development of the
bioeconomy.
In addition, there are two specific recommendations in relation to the field
production of platform chemicals/biopolymers and the opportunity for value added
co-product manufacture in energy crops. The first concerns set aside: this should be
reconsidered in the next round of CAP reform. The second concerns the risk that
permanent crops used for the non-energy bioeconomy will not be eligible for the
single farm payment. This is an urgent issue for consideration by the European
Commission.
1
1 INTRODUCTION
This is the second report of the biopolymer flagship for the EPOBIO project. The
aim of this report is an analysis of the suitability of selected crops to serve as
feedstock for the production of platform chemicals and biopolymers in 10/15 to 20
years. Most applications in this theme are in an early stage of development
necessitating a longer lead-time to market. The selection of crops that have been
analysed in this report is based on technical, environmental and economic criteria
discussed at the first EPOBIO Workshop in 2006 and finalised in subsequent
discussions with Consortium partners.
Our analysis focuses on the potential for new bulk platform chemicals and
biopolymers to be produced in planta as potential new industrial crop platforms for
Europe. Thus, existing large-scale commodity products such as starch, cellulose
and sucrose are not considered. Similarly, chemical or bioreactor-based
conversions of biomass post-harvest for the production of new plant bio-based
products are also not the focus of this study and plant oils are analysed in an
accompanying EPOBIO report [49]. Cell walls for biorefining purposes are analysed
in another accompanying EPOBIO report [180].
At present, most of the work on the production of biopolymers and chemicals in field
crops is only at the concept or early research stage of development since these bulk
products are typically manufactured via a microbial fermentation route due to the
current economics of the process. This study explores the future potential for field
production in three crops - sugar beet, tobacco and Miscanthus as well as
interesting developments in related species, such as, for example, in the case of
Miscanthus, the related grasses sugar cane. For tobacco, related Nicotiana species
or hybrids that may be better suited for biomass, chemicals and biopolymer
production than Nicotiana tabacum L. are discussed as well.
2
1.1 Platform chemicals and polymers from renewable materials
The expectation that petrochemical products will become scarce in the next
decades has fostered research and thinking on the potential of using crops to
produce energy (heat, electricity), fuels, chemicals and polymers [224, 241, 265].
Whilst most platform chemicals and polymers are derived from petroleum, a rapidly
growing feedstock is derived from renewable resources. Typical agricultural
products such as sugars, starch, cellulose, plant oils, and fibres are converted to
base- and specialty chemicals, polymers and materials using chemical methods or
biotechnology and fermentation. The latter routes, labelled as white or industrial
biotechnology, are rapidly gaining ground in the chemical industry, and are
expected to take a significant share of the chemicals and polymers market if oil
prices remains high and sugar prices do not become excessively elevated [196]. It
is important to note that currently, this increasing use of industrial biotechnology is
based on traditional crops and agriculture. As yet, industrial biotechnology is
relatively expensive due to the need for large capital investments in fermentation
equipment and process control, hindering take up of renewable feedstocks. The
question posed in this EPOBIO report is whether it is feasible both commercially
and from a sustainability perspective to produce platform chemicals and polymers
directly in large-scale field cultivation, thereby omitting the need for
(bio)conversions.
1.2 Production of platform chemicals and polymers in planta
It is a generally held opinion that the use of dedicated non-food crops bypasses the
issue of co-existence of industrial crops and food crops. This is particularly relevant
if the industrial crops are constructed via a genetic modification route (GM). Plants
may be optimised for the production of a specific chemical or biopolymer, or
cultivated principally to produce biomass with additional useful co-products.
Current attention for climate change (leading to levies on CO2 production) and the
peak oil concept (further increasing petroleum prices) lead to a rapidly increasing
3
market demand for biofuels. Now that the drawbacks of first generation biofuels are
increasingly recognized (increasing food-prices, unfavourable energy and CO2-
balance), the development of new routes becomes pressing. Thus, optimisation of
sustainable biomass (and biofuel) yield and efficient conversion to useful
compounds now is the primary goal (see the EPOBIO report on cell walls for
biorefining [180]). A secondary goal is the development of valuable co-products
through the introduction of genes and pathways specifying the production of
biopolymers or chemicals. In these applications, there should be no compromise of
agronomic properties, yield, processing, and safety of the crop and these
considerations clearly place severe constraints on the options. For example,
compounds that have some toxicity to the plant even when sequestered to specific
cell compartments can be expected to reduce yields, whilst other compounds such
as those that are not easily extracted may interfere with processing post-harvest.
The critical general question is whether the production of biopolymers or chemicals
in plants can compete with established and future fermentation methods or
chemical synthesis from petroleum or biomass. In theory, production in transgenic
plants is more direct and involves fewer steps than the use of an industrial
biotechnology route: the target compound is produced in and extracted directly from
the crop, whilst the fermentation route involves additional steps after extraction of
the sugar, starch or plant oil from the plant. However, whilst all the steps in the
fermentation route are based on proven and existing technology, many steps in the
use of field crops for chemical production are not established.
Whilst it is clear that industrial advances and low petroleum prices over the last
century have led to the replacement of agricultural by petrochemical feedstocks
derived from fossil reserves, there remain many desirable materials derived from
plants [241]. Certain products have become the basis for large global industries
(sucrose, starch, cellulose, plant oils, proteins, natural rubber, cotton, linseed oil,
cork), whilst other industries using plant-based products are smaller scale or
manufacture niche products.
4
Biotechnology now offers potential new routes to widen the range of industrial
products that can be made in field crops. Many of these applications are in the
concept stage with considerable research in progress for the development of
transgenic plants manufacturing commercial products such as industrial enzymes
and proteins for diagnostic and therapeutic purposes, modified plant oils and fatty
acids for applications such as paints and lubricants, biopolymers to replace
petrochemical plastics and specialty chemicals, such as pigments, flavours and
fragrances [13].
Many such studies have failed benchmarks because of effects such as, for
example, negative impacts on plant health and the inability to change metabolic flux
at will. Nevertheless, there are examples of success in transgenic applications.
Sugar crops have been modified to produce useful chemicals or polymers that can
be derived from sugar building blocks via only limited a few enzymatic steps. In this
context sugar manipulation in sugarcane (a close relative of Miscanthus; chapter 5)
has led to transgenic plants in which 60% of the soluble sugar is sorbitol [92], and
plants producing isomaltulose in addition to sucrose at unchanged concentrations,
doubling the total sugar concentration in the juice [274]. Sugar cane was also
altered to produce p-hydroxybenzoate. Here, the highest concentration detected
was 7.3 % in the leaf [171].
The production of a new polymer in a plant has been demonstrated using a
transgenic sugar beet [228]. The fructosyltransferase of Helianthus tuberosus
expressed in sugar beet converts sucrose into low-molecular-weight fructans, a low
calorific sweetener. An astonishing 40% of the taproot dry weight was found to
consist of fructans, without any detriment to the host plant (Chapter 3). Similarly,
tobacco has been engineered to produce up to 25% p-hydroxybenzoate [260], and
smaller amounts of other chemical compounds (Chapter 4).
Applications involving oilseed crops can include related compounds such as wax
esters [180], designer oils [4, 241] or alkanes [269]. Some of these are considered
5
in the accompanying EPOBIO report on plant oils [49]. Oil crops could also be used
for the production of hydrophobic polymers such as polyhydroxyalkanoates [197].
The potential risk of producing industrial chemicals and biopolymers in plants must
also be considered, as has been done in the 2005 study by the Office for
Technology Assessment of the German Parliament [219]. One aspect of this study
is new risks associated to feedstock crops. For example, many of the envisaged
products are potential anti-nutrients. Thus, dual use of the crop for chemical
feedstock production and animal feed is considered unlikely; the remaining biomass
is only suited for bioenergy production. In the same study, the use of plants for the
production of industrial compounds is judged to be relatively distant, even though
plants containing modified oils (rape containing lauric acid and soy with an
increased oil-content) and starch (high-amylose and amylose-free) have already
been commercialized. In part, the development of industrial crops for chemicals and
biopolymers is difficult to know, since the R&D is mainly undertaken within industry
and is not in the public domain. It is also the case that developments in this
industrial sector would seem to be much slower than those predicted in the past, for
example, it has been assumed for many years that bioplastics would be developed
in crops and as yet this has not occurred in Europe. Also, yields are often lower and
heterologously expressed genes function less well than expected. It is also
concluded that a single-use crop is not likely to be successful due to relatively low
yields, and that co-production with biofuels is a viable strategy [219].
On the economical potential for chemical production in crop plants the same study
[219] suggests that the production of new compounds using this plant route may
well be successful, but without the general cost advantages that have been
predicted earlier. The study further states that the success of the in planta route will
always be determined by parallel advances of competing production systems: such
as white biotechnology and novel eukaryotic cell culture systems, the development
and cost of specific cultivation schemes (e.g. all-greenhouse in the case of plant-
made pharmaceuticals), and risk management [219].
6
The fact that the in planta production of chemicals and biopolymers requires GM-
technology is of great significance. Although the technical possibilities have been
demonstrated there are still relatively few GM-crops in field. This is so because the
process of developing a transgenic crop is much more complicated and costly than
is generally realized (Figure 1). Assuming that all technical issues can be resolved,
regulatory issues have to be faced. Toxicity testing of the plant alone can cost in
excess of U$ 500�000. Regulatory clearance, clearance for import, and variety
registration, are difficult steps, and may have to be repeated from country to
country. For a single country the costs may be in the range of U$ 2 � 3 million, while
global registration will cost upward from U$ 5 million. In total, discovery, R&D,
breeding, production, admission and other regulatory matters may cost several tens
of millions of US Dollars [72].
Time-line in years
1 2 3 4 5 6 7 8 9 10 11 12
gene discovery
transformation, GM-production
greenhouse testing, molecularcharacterisation, processing
proof of concept in the fieldlarge scale processing testing
selection of suitable variant
regulatory clearance
introgression, testing in differentgenetic backgrounds
breeding, variety registration
regulatory clearance(toxicity, etc.)
scale-up, seed production
commercial sales
key milestones
Figure 1 Approximate time-lines for the development of a new transgenic
crop, from gene discovery to commercial seed sales. The time-lines can vary depending on speed of success, and regulatory requirements (from [72]).
7
In general, new transgenic crops must offer a sufficiently large advantage to farmers
and a substantial assured market to make uptake possible. Equally there must be
substantial public acceptance and encouragement for products manufactured by
GM-crops to give confidence to manufacturers to enter the lengthy process of
transgenic crop development. For chemicals and polymers, this is only possible
either for bulk or for mid to high-price compounds. In the case of bulk compounds, it
must be realized that GM-crops would have to compete with non-GM biomass and
biofuels crops. The situation can be compared with the development of herbicide-
tolerant crops, which have clear benefits to growers but are still not widely accepted
by the general public nor planted extensively in European member states [72].
1.4 Selecting crops for the production of platform chemicals and biopolymers
Most significantly, for development and uptake by industry, the commodity
production of platform chemicals and biopolymers in field crops should compete
economically with petrochemical feedstocks. Whilst dedicated new industrial crop
platforms for commodity chemicals might prove too expensive, there is every
reason to predict that feedstocks will become co-products of biomass production.
In this scenario, the crops for production of platform chemicals and biopolymers are
likely to have the same features as those selected for analysis in the cell wall for
biorefining flagship [180]. Thus, these crops should have:
! A high biomass yield
! Cell walls amenable to energy efficient bioconversions
In addition to those features specific for chemical/biopolymer production, namely:
! Lack of negative impact on the plant of producing high yields of a novel
industrial product
• Knowledge of metabolic pathways and metabolic engineering
• Amenable to nuclear transformation, but also preferably to routine methods
for chloroplast transformation
8
Generic features recommended for all industrial crop platforms irrespective of
specific uses include:
! Low input requirements in terms of fertiliser, pesticide and water usage
! Low impact on biodiversity, soil and water quality
! Efficient land use with high carbon sequestration rate
! High safety in terms of gene flow
! Ability to grow on marginal land and those cultivation areas that do not
compete with land use for arable food crops
! Low investment costs in terms of labour, machinery and energy
These issues were considered at the EPOBIO Workshop in 2006 and in subsequent
discussions amongst the Consortium partners. As a consequence, this analysis
includes the use of a dedicated biomass crop (Miscanthus), an established crop that
is already processed in an industrial setting (sugar beet), and a crop that is highly
amenable to GM modification for high yields of novel products (tobacco).
9
2 THE POLICY CONTEXT FOR THE BIOECONOMY 2.1 Introduction
An opportunity exists to build a bioeconomy delivering sustainable economic growth
with job creation and social cohesion as key outcomes. Creating such a bio-
economy involves the substitution of fossil materials with renewable carbon. As a
consequence of increasing the use of renewable resources for industrial feedstocks
and for energy, the bioeconomy will bring benefits in a number of areas. Some
examples of these benefits are:
! Reduced dependence on imported fossil oil.
! Reductions in greenhouse gas emissions.
! Building on the existing innovation base to support new developments.
! A bio-industry that is globally competitive.
! The development of processes that use biotechnology to reduce energy
consumption and the use of non-renewable materials.
! Job and wealth creation.
! The development of new, renewable materials.
! New markets for the agriculture and forestry sectors, including access to
high-value markets.
! Underpinning a sustainable rural economy and infrastructure.
! Sustainable development along the supply chain from feedstocks to products
and their end-of-life disposal.
In order to deliver these benefits it will be necessary to address a number of key
challenges. Firstly, the potential of plant science to help deliver the bioeconomy is
not well understood. This generally low level of awareness exists amongst
politicians, policy makers, the general public and those likely to benefit directly, such
as farmers and foresters. Secondly, large companies are reluctant to move away
from production systems that are based on fossil oil. Feedstock costs will inevitably
be a driver but there is also lack of experience of, and nervousness about, supply
chains that originate in the agriculture and forestry sectors. There is also an
10
absence of validated techniques for the measurement of sustainability benefits.
Finally, building the bioeconomy requires the development of policies and a
regulatory framework that recognise the linkages between a range of issues which
include bio-resources, renewable feedstocks for energy and manufacturing,
sustainable growth and employment, sustainable communities, climate change and
other environmental issues and impacts.
2.2 Biorefineries and the bioeconomy
From a policy and regulatory perspective, the development of efficient and cost
effective biorefineries is important for a number of reasons. Biorefineries are a key
element in the bioeconomy, delivering renewable and sustainable products able to
compete with existing fossil-derived products. Biorefineries already make a positive
contribution to the delivery of international targets and governmental commitments
for reductions in greenhouse gas emissions, whilst also addressing energy supply
issues. Innovation directed to the development of new generations of more efficient
biorefineries will deliver a major improvement in the level of the greenhouse gas
emission reductions achieved.
Advances in plant science and biotechnology will underpin the future development
of biorefineries that will support more diversified agriculture, forestry and industrial
production systems that are more sustainable and deliver economic and societal
advantages. Alternatives to food production will contribute to the redevelopment of
rural areas.
The increasing concern about the environmental impact of the expansion of oil
palm, soybean and sugar cane cultivation for biofuels feedstocks, leading to
deforestation in Indonesia, Malaysia and Brazil can be addressed through the
development of new generations of biorefineries. The future development of more
efficient, second and third generation lignocellulosic biorefineries in Europe and the
US affords the potential to track and evidence environmental impacts and benefits
and increase the efficiency of production of biofuels, and bio-based materials and
11
chemicals. In parallel, it should be possible to reduce dependence on imported
feedstocks and so help address environmental concerns about their production and
use.
2.3 Fossil oil and biofuels
The production of biofuels in biorefineries and reducing dependence on fossil
reserves is driven by a number of strategic imperatives including the price, finite
nature and security of supply of fossil oil. Other factors include the detrimental
environmental impact of fossil-derived fuels and mineral oils versus the renewable
and sustainable nature of plant-derived alternatives.
There are also important regulatory drivers such as the indicative target in the EU of
5.75% biofuels by 2010, a target that has now been extended to 10% by 2020
�subject to biofuels becoming commercially available�. In the US, policy initiatives
include the Energy Action Plan, mandating an increase in the use of bioethanol and
biodiesel, and the Advanced Energy Initiative promoting the development of
practical and competitive methods for the production of bioethanol from
lignocellulose.
The expectation is that the biofuel industry will develop to significant size,
consuming a significant proportion of biomass feedstocks. One concern is that the
development of this industry depends on subsidising the product at the point of
purchase. In addition, the environmental deliverables need to be compared to other
potential options to, for example, reduce greenhouse gas emissions. This requires
that a holistic view of the bioeconomy be taken, rather than feedstocks, products
and markets being developed in isolation.
12
2.4 Common Agricultural Policy (CAP)
For the successful development of the bioeconomy it is essential that there is a
robust agriculture sector that can provide a reliable source of feedstocks and deliver
consistency of supply, price and quality. The 2003 reforms of the CAP brought a
significant simplification, introducing a payments system of a single support
payment made on a per hectare basis. Reforms were extended to the
Mediterranean crop regimes (tobacco, cotton and olive oil) and to hops in 2004,
which broke the link between subsidy and production and brought a new focus on
the market. Linked to actions designed to deliver sustainable farming strategies,
these reforms provide a sound basis for farmers to take advantage of a new
flexibility to innovate and seek out new markets. New income opportunities in
farming are linked to the potential for diversification in agriculture and the new
commercial markets of the bioeconomy will help farming, encourage sustainability
and underpin the wider rural economy and its infrastructure.
Within the single farm payment scheme, land used for the cultivation of permanent
crops (non-rotational crops that occupy the land for five years or longer and yield
repeated harvests) is not eligible. Permanent crops include, for example, short
rotation coppice and Miscanthus. However, short rotation coppice and Miscanthus
can be grown on non set-aside land and the single payment received if the energy
crop aid is also claimed. Any permanent crop or tree species used for non-energy,
non-food applications would not be eligible for the EU single farm payment scheme
unless grown on set-aside land. In this way, permanent crops grown as feedstocks
for non-energy products are disadvantaged compared to those grown for energy
products.
The retention of compulsory set-aside and the requirement to withdraw land from
agricultural production, has maintained an opportunity and an incentive to produce
feedstocks for biofuels and biorenewables. Under the set-aside rules, the
production of crops for specified non-food uses is allowed subject to certain
conditions, including a requirement for contracts and the payment of securities.
13
Currently 8% of land must be set-aside. There is no guarantee that set-aside will be
retained in any future review of the CAP and it could be said that it has no place in a
market-focussed CAP. This inevitably builds uncertainty into the future production
of bio-based feedstocks.
There is an energy crop aid of �45 per hectare for crops grown on non set-aside
land. Originally this was paid for a maximum guaranteed area of 1.5m hectares of
land, but has now been increased to 2m hectares to extend availability to the newer
Member States of the EU. If this ceiling is breached the aid will be reduced pro
rata.
Multiannual crops generally have considerably higher establishment costs than
annual crops. Support for establishment costs is possible under EU rural
development regulations. Regulations also allow Member States to grant national
aid of up to 50 per cent of the costs of establishing multiannual crops.
A simplification exercise is currently underway for the CAP. This began in
December 2006 with a proposal to establish a single Common Market Organisation
(CMO) for all agricultural products, to replace the existing 21 CMOs. The aim is to
provide a single set of harmonised rules in the classic areas of market policy, such
as intervention, private storage, import tariff, quotas, export refunds, safeguard
measures, promotion of agricultural products, state aid rules and communication
and reporting of data. The substance of existing rules and mechanisms will not
change. It is expected that the simplification will enter into force in 2008.
It is essential that policy frameworks are well coordinated. Agriculture and forestry
have a critical role to play but the bioeconomy impacts on over fifteen policy areas
in the EU and on the work of 10 of the EU�s Councils. There is a crucial need to
look holistically at the development of the bioeconomy, from feedstock production
through to products and their end of life disposal. The full range of feedstock
supplies needs to be considered, including material from agriculture, forestry and
from the waste sector. There must also be an overview of the full range of industrial
14
developments that are being promoted including biomaterials, biofuels and other
forms of renewable energy from biomass.
2.5 Use of genetically modified plants
The implications for the use of a genetically modified plant, the impact of current
GMO regulations in Europe and the associated substantial regulatory compliance
costs have to be considered. Small and medium sized enterprises are unlikely to be
able to bear the costs associated with these issues and so future exploitation is
likely to be undertaken only by multinationals. Taken together, these constraints
have the potential to limit development in Europe and lead to a continuing
dependence on imported fossil oil and a continuing loss of competitive advantage to
other countries and regions where the cultivation of genetically modified crops is not
constrained.
The risks associated with the use of a genetically modified crop can be mitigated in
a number of ways. The use of a crop that cannot be used for food or feed is
important. This is considered essential from a regulatory perspective, given that the
infrastructure in agriculture cannot ensure �fail-safe� separation of different
varieties/traits in the same crop species. However, the use of a non-food crop can
have negative consequences since, for example, oil crops such as Crambe have
not been optimised for mainstream agriculture and their oil yield needs to be
improved.
Risks can also be mitigated by the choice of a crop for which inter-species crosses
with the closest-related species give sterile offspring. A third means of risk
mitigation is the adoption of the same identity preservation practices for the
cultivation of non-food GM crops as those already in place for the cultivation of GM
food crops.
15
2.6 Land use and availability
There are a number of studies, relating to the future development of the
bioeconomy that address land use and land availability issues. But, to date, they
have done so in isolation either looking at feedstocks for biofuels, feedstocks for
other forms of energy such as heat or electricity, or feedstocks for non-energy
bioproducts. Studies have not generally had regard to the totality of potential
development. There will be an essential need to balance food security and supply
with the production of raw materials for the bioeconomy as a whole. The
development of the bioeconomy means that there are key issues and
consequences:
! Increasing demand for productive land � land becomes scarce.
! Increasing questions about land use � security of supply.
! Increasing competition for land � range of different crops.
! Increasing use of marginal land.
Land use and availability issues will need to be addressed across the whole
bioeconomy landscape in the near future.
2.7 Climate change
Climate change is regarded as one of the greatest environmental, social and
economic threats facing the planet. There are international efforts to combat
climate change and the two major treaties addressing this issue are the United
Nations Framework Convention on Climate Change and its Kyoto Protocol.
The Convention on Climate Change sets an overall framework for
intergovernmental efforts to tackle the challenge posed by climate change. It
recognises that the climate system is a shared resource whose stability can be
affected by industrial and other emissions of carbon dioxide and by other
greenhouse gases. The Kyoto Protocol to the Convention assigns mandatory
targets for the reduction of greenhouse gas emissions in signatory nations.
16
Climate change presents an opportunity for the bioeconomy, through the use of
plants and forest materials as feedstocks, to displace fossil alternatives and so
reduce greenhouse gas emissions. Biofuels, for example, are more sustainable
and environmentally friendly because of the reiterative cycles of burning, followed
by carbon fixation by plants, followed by burning of biofuels. Bio-renewables are a
sustainable means of providing the essential products needed by society.
The potential of green plants to use solar energy and manufacture raw material
feedstocks offers a way to help address these issues and to deliver the sustainable
development needed to underpin future societal needs and demands. Crops
provide a sustainable and clean technology with the potential for high capacity and
the ability to produce feedstocks for energy or complex chemicals, yielding multiple
products from a single crop. Agriculture, horticulture, forestry and aquaculture can
provide products for all aspects of our lives including food, feed, medicines,
chemicals and materials. Non-food applications of crops and the potential for
renewable energy are also increasingly important.
Climate change does, on the other hand, also give rise to real concern in respect of
the sustainable development of agriculture and forestry globally. Temperature
changes, water availability and extreme weather conditions are among the issues
that will impact on agriculture in the years to come. There will be an impact on
crops in terms of types, locations and yields and a potential loss of production
potential in some geographic regions. Crop patterns and management practices
will need to adapt to new scenarios. This will raise serious challenges for
bioeconomy feedstocks and for agricultural incomes.
2.8 Sustainable development
There is a growing realisation that our current model of development is
unsustainable and that the increasing burden we are placing on the water, land and
air resources and on the environmental systems of our planet cannot continue.
Sustainable development is about meeting the needs of present generations without
17
jeopardising the needs of future generations. It involves a better quality of life for
everyone, now and for generations to come. It offers a vision of progress that
integrates immediate and longer-term needs, local and global needs, and regards
social, economic and environmental needs as inseparable and interdependent
components of human progress.
The EU sustainable development strategy sets overall objectives, targets and
concrete actions for seven key priority challenges for the coming period until 2010.
Of the seven areas, five are relevant to EPOBIO and the bioeconomy. They are:
! Climate change and clean energy
! Sustainable transport
! Sustainable production and consumption
! Better management of natural resources
! Fighting global poverty
The use of crops for the production of bioproducts has the potential to help deliver
these elements of the sustainable development agenda. In this as well as other
policy areas, the absence of validated techniques for the measurement of
sustainability benefits will need to be addressed so that the gains can be evidenced.
2.9 Developing countries
Developing countries have the potential to share in the expansion of the global bio-
economy, and its commercial returns, through the production of feedstocks and
their processing. This is an innovation that will be market-led and could develop an
industrial base, trade and the underpinning agricultural production. The
development of crop production and processing in developing countries has the
potential to deliver wealth creation and access to trade.
The importance of the agriculture sector in developing countries means that the
expansion of the agro-industrial sector would bring an opportunity to reduce poverty
in a sustainable way. Biorefineries and the production of bioproducts in developing
18
countries could readily deliver social and economic benefits through the production
of biofuels and energy for local use, integrated with bioproducts for export.
These productive activities, based on market-led innovation, developing technology
and innovation, would provide access to new and growing markets. Poverty
reduction through the revitalisation of the agro-industrial sector would be a tangible
outcome of the production of feedstocks and the development of bioproducts in
developing countries.
2.10 Industrial competitiveness
Industrial competitiveness in the bioeconomy depends on a number of factors.
There is a need for a policy framework that is integrated and coordinated. The
range of feedstock and their applications means that the bioeconomy is relevant to
a large number of policies from agriculture through to trade and waste
management. Policy developments should be considered for their impact on the
bioeconomy and its future expansion. Existing regulatory barriers to moving
traditional industry to a more sustainable bio-based approach will need to be
removed, for example in the approval of bio-based products that replace existing
chemical alternatives.
Support for innovation is an essential underpinning for the development of the
bioeconomy. Research funding, industrial engagement, the participation of small
and medium sized enterprises and technology transfer are key elements.
Political and policy initiatives, such as future reforms of the Common Agricultural
Policy, can set the framework within which the future production of feedstocks for
the bioeconomy can take place.
The development of a competitive bioeconomy will deliver tangible outcomes in a
wide number of sectors of industry. As well as access to markets and trade,
outcomes will include job creation in agriculture, forestry, the transport sector and
manufacturing. Support for rural communities and the rural infrastructure will be
part of this.
19
2.11 Strategic conclusions and recommendations
There are key elements that need to be in place to set a policy and regulatory
framework for the development of the bioeconomy:
1. Policies must be coherent, integrated and coordinated.
Integration in Brussels and Member States is essential to develop a policy
framework that will support the bioeconomy. As the bioeconomy represents a
potentially huge strategic development consideration should be given to applying a
�bioeconomy test� to policies in development, in the same way that policies are
assessed for their sustainable development impacts.
2. Innovation in plant and industrial biotechnology should be supported.
Clear research objectives and a framework to achieve them are essential. An
adequate level of targeted funding, selecting those novel and innovative processes
and products likely to achieve success in the market place and deliver
environmental benefit, should be an element of this. There is also a clear and
present need to establish the knowledge base in the post-genomic era that will
allow more rational design of crop plants that are tailored to produce high value
bioproducts and biofuels. This is a longer-term objective that should be developed
based on clear marketing potential and impact.
3. Policies should support development of the whole supply chain.
This will need to consider feedstock supply, processing and the production of
bioproducts. There is a need to both stimulate the market side and build on the
foundation of the Common Agricultural Policy, which has moved from production
subsidy to market-orientated developments. Financing along the supply chain
needs to be considered as one aspect of feedstock supply.
20
4. A communication strategy is essential.
The acute lack of awareness of the bioeconomy and the potential of biotechnology
at all levels in society must be addressed by a strategic communications campaign
designed to raise awareness and create an informed acceptance of bioproducts.
This will need to explain the benefits of the processes and products delivered by the
bioeconomy.
5. Pilot projects have a role to play.
The establishment of proof of concept and testing under industrial conditions is a
key step in moving research into product development. Scale-up during the
research phase can develop and test industrial processes and also help to develop
stronger co-operation between industrialists and academics.
6. Measurable sustainability indicators should be developed.
The absence of validated techniques for the measurement of sustainability benefits
needs to be addressed. This is important as these gains need be evidenced to
enable all stakeholders to understand the rationale for the development of the
bioeconomy.
2.12 Specific conclusions and recommendations for the biopolymers and platform chemicals crop platform We make the assumption that the market should lead the expansion of the
bioeconomy and biorefining. We consider that subsidies � which are inevitably
unsustainable, distort the economics of the market place and can be removed at
any time � should not be a feature of the development of this sector.
In this context, the 2003 reforms of the Common Agricultural Policy provide a sound
basis for the development of crop platforms to provide feedstocks for activities led
21
by the market. Of the crops considered in this report, sugar beet and tobacco can
be cultivated on both non set-aside and set-aside land and can access the single
payment. But the retention of set-aside as a mechanism to influence production is
inconsistent with this market-led approach and the cultivation of non-food crops on
set-aside land does also involve the producer in a significant degree of bureaucracy
and cost. We recommend that the future of set-aside be reconsidered in the next
round of CAP reform.
In the case of Miscanthus, this and other permanent crops are disadvantaged when
grown for the production of feedstocks for the non-energy bioeconomy. The current
energy crops aid targets only those crops destined for the energy sector of the
bioeconomy. Also, land used for the cultivation of permanent crops is ineligible for
the single farm payment scheme, unless those crops are grown for an energy
purpose and the energy crops aid also claimed. In this way, the energy component
of the bioeconomy is given more favourable treatment within the CAP than the non-
energy element, crops for the latter being denied access to the single farm payment
and disadvantaged in a competitive market by the energy crops aid. As permanent
crops have the potential to form a significant part of the feedstocks for the
bioeconomy, we recommend that the European Commission review the current
disadvantages faced by permanent crops grown for the non-energy bioeconomy.
22
3 SUGAR BEET (BETA VULGARIS)
3.1 Introduction
Most plants produce sucrose as a temporary carbon-storage product, and convert it
to starch for long-term storage. However, two species, sugar beet (Beta vulgaris L.)
and sugar cane (Saccharum officianarum L.) accumulate exceptional amounts of
sucrose. Together they account for more than 90% of the world's sugar trade, 11%
of the world's food supply, and 0.2% of all the carbon fixed via photosynthesis by
the world's crops each year. World sucrose production in 2006/2007 is estimated to
be 155 million tons. In 1998/1999, 28.5% was derived from sugar beet [10], The EU
harvest of sugar beet in 2005 amounted to 131 million tons (Table 1). Sugar beet is
the most productive conventional crop grown in Europe: in addition to the 12 t ha-1
sucrose, 3 t ha-1 pulp (dry weight) and 5 t ha-1 leaf (dry weight) can be obtained
each year.
Table 1 Top ten Sugar Beet Producers – 2005 (Million tons)
France 29 Germany 25 United States 25 Russia 22 Ukraine 16 Turkey 14 Italy 12 Poland 11 United Kingdom 8 Spain 7World total 242World growth area 8.2 million haEU total 131Source: http://faostat.fao.org
23
While sugar cane has been used for thousands of years as a source of sugar [127],
sugar beet is a relatively new crop. In 1747 a German chemist, Andreas Marggraf,
found that crystals formed after a crude extraction from pulverized fodder beets
were identical with sugar cane crystals. His student, Karl Achard, made the first
selections of higher sugar type beets, and developed cultivation and processing
methods. The imposition of the blockade of the continent during the Napoleonic
wars limited access to cane sugar in Europe and stimulated a plant breeding
programme and the construction of many sugar beet processing factories. After the
French defeat at Waterloo, the nascent sugar beet industry declined again.
Nevertheless, modern sugar beet selection, and the feasibility of a processing
industry had been demonstrated.
Over the following two centuries, a sugar beet based industry slowly developed in
the Europe, North America, and Russia. Almost everywhere this was made possible
only by taxing cane sugar imports. Restrictions on imports by the EU combined with
subsidies for farmers and the processing industry have kept the industry viable in
the past decades. However, new EU policies make sugar production in the EU
much less attractive. In November 2005, the Agriculture Council reached agreement
in the first major reform of the EU sugar regime since it was introduced nearly 40
years before, bringing it into line with the rest of the reformed Common Agricultural
Policy. EU sugar prices are being cut by 36% over 4 years alongside a voluntary
restructuring scheme aimed at reducing production by around 6 million tonnes in the
same period. This will enable the EU to comply with the recent World Trade
Organisation ruling limiting subsidised exports and fulfil its existing commitments on
preferential imports from ACP and LDC countries without imposing new restrictions.
It aims to ensure a long-term sustainable future for sugar production in the EU,
enhance the competitiveness and market-orientation of the sector and strengthen
the position of the EU in world trade talks. Sugar beet is an eligible crop under the
EU single payment scheme and can be grown both on set-aside and on other
arable land. Production on set-aside is subject to conditions, including a
requirement for contracts and payment of securities.
24
In the Netherlands, the CAP-reform reduces income from sugar beet cultivation
from � 1350 ha-1 to � 550 [203], which is comparable to the net profit from maize,
wheat and barley. On the assumption that the average net profit should be � 800,
with an absolute minimum of � 600, sugar beet becomes a clearly less attractive
crop. According to the FAO, sugar production in developed countries is expected to
decrease by 9.1% in 2006/2007, down to 39.1 million tonnes. Output in the EU for
2006/2007 is estimated to have fallen by 23%, from 21.4 million tonnes in
2005/2006 to 16.5 million tonnes in 2006/2007, reflecting the adjustment process
begun in July 2006, under the European Union sugar policy reform [12].
The recent closing of the British Sugar factory in York, England and the
diversification of the British Sugar plant at Wissington into biofuels illustrate the
consequences. The processing industry and farmers alike are interested in ways to
maintain sugar beet as an economically viable crop, due to its indisputable values
as a high biomass crop, a useful break crop, and a crop that is highly profitable for
the farmer.
3.2 Beet processing
3.2.1 Sugar beet processing
Companies that process sugar beet currently have strong interest and considerable
influence in all aspects of beet production. This influence ranges from the area
planted through to sugar marketing. Without a processing facility, sugar beet
currently is of little value. Similarly, once a sugar factory is constructed, a reliable
supply of beets is essential to the company. The supply-chain linking grower to user
is therefore locked. These relationships have led to a strong vertically integration,
and a cooperative relationship between growers and sugar beet processing
companies. In the United States and mainland Europe, farmers often own the
processing factories as cooperatives. In other regions, individual growers and the
processing company set up contracts specifying the area that is planted, details on
the delivery of beet roots and payment arrangements. The growing areas and price
25
have been determined by quota. This will irreversibly change as a result of the CAP
reform.
Table 2 Major European beet sugar producers
1 Südzucker Germany 23.5 % 2 Tereos France 9.4 % 3 Nordzucker Germany 8.8 % 4 British Sugar Britain 7.6 % 5 Danisco Denmark 6.6 % 6 Pfeifer & Langen Germany 6.2 % 7 Ebro Puleva Spain 4.5 % 8 Eridania Italy 3.6 % 9 Italia Zuccheri Italy 3.4 % 10 Cosun Netherlands 3.4 %
In % of European quota. Total production: 17,441,000 tons
(From the annual report of Tereos 2004/2005)
The strong vertical integration means that product diversification depends on the
capabilities, structure, and interests of the processing industry (Table 2). Factors
such as harvesting campaign, storage, and integration of new processing steps in
existing facilities can greatly influence opportunities for innovation and change. It is
essential to consider these issues in parallel to the design of any new co-products
engineered into sugar beet. It is clear that the attitudes and expectation of the sugar
processing companies will be all-important. In this context the recent alliance of
British Sugar with BP and DuPont to develop new biofuels from a sugar-based
feedstock is significant and may flag a new approach in the industry with
consequences for beet as an industrial crop platform.
3.2.2 Conventional processing steps used for sugar crystallisation
Judging whether new co-products engineered into beet can be recovered efficiently,
it is necessary to consider practices in current or future beet processing (Figure 2).
26
Thorough overviews of methods, procedures, and technology can be found in the
Beet-sugar handbook, by Mosen Asadi [17], and Sugar Beet, edited by Philipp
Draycott [77].
In order to guarantee a continuous beet supply for processing, beets are usually
stored in field clamps and/or at the factory yard. Maximum storage and thus the
possible processing period depend on climate conditions, and range from a few
weeks in the Mediterranean to up to several months in Scandinavia. Improper or
overlong storage can cause sugar and mass losses due to beet respiration and
decay by microorganisms. Part of the sugar is converted to invert sugar, which
further converted to lactic acid.
Sugar beets are delivered to the processing facility by trucks and piled in the factory
yard or dumped directly into wet hoppers. From there the beets float on the water
into the factory in a flume, passing through rock-catchers for the removal of any
rocks, mud or sand, and then through another section for the removal of trash,
weeds, and leaves. Then they are washed in a beet washer and a roller-spray table.
Next, slicers cut the beets into long noodle-like pieces called cossettes, which are
conveyed into a scalding tank. Here the sugar dissolves in hot water in a continuous
counter-flow set-up and leaves in the form of "raw juice". The cossettes leave the
extractor as beet pulp and are moved on to the pulp dryer (see section 3.3.3).
To remove impurities and other non-sugars the raw juice is subjected to various
stages of purification and filtration. After heating in the raw juice heaters it is
pumped to the first carbonation station where milk of lime and CO2 from the limekiln
are added (first carte tank). The carbonated juice flows onto the Dorr thickener
where the precipitate formed in the juice of milk of lime and carbon dioxide is
separated. The resulting clear juice is sent to heaters and then on to the second
carbonation station (second carte tank). The sludge recovered from the thickener is
washed to recover residual sugar before transfer to a holding pond. The filtrate and
the wash water from the drum filters are called "sweetwater", which goes to the
limekiln to be mixed with burned lime. Any excess goes to the first carte tank.
27
In the second carte tank, the clear sugar juice is mixed again with CO2 gas to obtain
the proper pH. Precipitates are again removed by filters. In a third saturation step,
the juice is mixed with SO2 gas to inhibit colour forming reactions and set the final
pH for sugar end liquors. The so-called thin juice then moves on to the evaporator
supply tank, through heaters, and then to the evaporation station. This consists of
five stages working at a temperature range of 98-130°C at different pressures in
which steam heat removes excess water. This process concentrates the juice from
about 15 percent dry weight to 65 percent to 70 percent. The resulting thick juice
goes to the high melter station, where it is used to dilute and melt the high raw
sugar. The standard liquor moves on to the white pan to be boiled and crystallized.
As so-called "white massecuite" or �crystal magma� it then drops into the white
mixer and subsequently into the white centrifuges. Here, the sugar crystals are
separated from the liquor containing sugar syrups and impurities. The crystals
remaining in the centrifugal basket are washed, dropped to a conveyor and moved
to the granulator for further drying and cooling. The finished sugar is then stored
and packaged. Several sugar containing side-streams containing impurities leave
the process as molasses (see section 2.3.3).
The above paragraphs describe the "straight-line" flow of the current processing
steps leading from sugar beet to crystalline beet sugar. Other "side-line" processes
recover more of the sugar from the side-streams by concentrating, purifying and
recycling steps. These technologies include the traditional Steffen process in which
sucrose is precipitated with CaO, and new methods, such a molasses desugaring
by chromatography (MDC), which increases the sugar yield from 80% to 90% [17].
While sugar beet processing has remained essentially the same from the early 20th
century, its present operation differs with more sophisticated equipment and
controls. In part these changes are due to higher standards for the finished product
for human food consumption, a greater focus on energy efficiency, and larger
factory capacity [17].
28
press water sliced beet water
wet pulp
pressed pulp raw juice
pellets thin juice
thick juice
sugar molasses
limeCO2
carbona-tionlime
animal feed food animal feedfermentation
fertiliser
extraction70�C, 100 min
juice purification75-95�C, 60 min
filtration
pellleting
65-75�C, 3-4 hcentrifugation
evaporation98-130�C, 100 min
evaporationcrystallisation
Figure 2 Principle steps of sugar beet processing and common product uses (adapted from [10]).
Sugar beet factories produce more waste than the combined output of sugar,
molasses and pulp. Some of these waste streams are already utilised [17], but
regulatory pressure forces companies to streamline their processes and convert
side streams to useful products. Although most of these opportunities may be in the
generation of biogas or re-use of waste-heat, more elaborate processing schemes
may also create opportunities for co-products engineered into the beet.
29
3.2.3 Side products of conventional sugar beet processing
Sugar beet processing yields a range of classical by-products that have found many
applications. These are molasses, beet pulp, and lime.
Lime
Carbonation lime leaving the process is used in agriculture as a fertiliser providing
calcium and a certain amount of plant nutrients such as N and P. It increases the
pH of the soil and thus improves soil structure. Some proteins specifically partition
to this fraction, allowing their easy separation from other beet components.
Molasses
Molasses are basically the remaining sucrose syrup that contains too high a
concentration of various impurities for sugar crystallization. Most of the molasses
(about 60%) is used in animal feed as feed ingredient, pelleting aid or ensiling
agent. Another 15% is used in fermentation (to obtain products such as yeast, citric
acid and alcohol). Other applications of molasses, e.g. as a source for single
substances such as betaine are currently of minor economic importance. Various
industrial purposes such as fuels, rubber, printing, chemical and construction
industries also consume minor amounts of molasses.
Although the main component of molasses is still sucrose, it contains many
additional and potentially valuable components such as mono- and
oligosaccharides, polyols, vitamins, uridine and other ribonucleosides, betaine,
amino acids such as glutamine, and organic acids such as lactic acid,
pyrrolidonecarboxylic acid, γ -aminobutyric acid, and D- and L-pyroglutamic acids.
As a group the amino acids are the most valuable component, while raffinose is the
most valuable single component. The current trend to molasses desugaring (MDC)
by conventional or simulated moving bed chromatography present interesting
opportunities to recover these compounds [99, 130, 255], and potential future
30
compounds engineered into beet (provided that these compounds do not interfere
with sugar crystallization, i.e. lower the technical quality: see section 3.2.4). Besides
sugar, the main products of MDC are betaine and a mixture named raffinate. This
consists of salts, protein, sucrose, raffinose and nitrogen compounds. Both are sold
as a valuable liquid feed supplement. The return on investment of the MDC process
depends mainly on the processing period in days per year and varies between 3.4
and 5.7 years [17].
Pulp
The wet pulp leaving the diffusers is transferred to the pressing section. Here, a
pulp press and dryer drums reduce the moisture content of the pulp to about 10
percent. In pellet mills, the pulp is converted into a solid form for easier handling.
Most of the pulp is marc (68%, pectin, cellulose, hemicellulose, lignin), sugars
(14%), protein (6%), and ashes (12%) (Figure 3). The energy content of dry pulp is
15 MJ kg-1 [264], however, the pulp has an even higher value as fodder and
provides a highly nutritious feed for livestock (cows, horses and pigs).
Figure 3 Beet pulp contains a range of potentially valuable compounds, including protein, pectin, sugar and sugar-polymers [21].
31
Heterologously produced biopolymers would end up in the pulp fraction in
conventional beet processing. The pulp could then be fractionated to obtain the new
product and other components of the pulp such as pectin and cellulose. Pulp has
been tested as a source of galacturonic acid (a building block for pharmaceuticals,
cosmetics and food ingredients) using a commercial enzyme mix [22]. Pulp may
also be fermented to bioethanol [75].
3.2.4 Biofuels and beet technical quality
Sugar and fodder beet are already used for the production of biofuels, and in 2004 1
million tons or 0.8% of sugar beet went toward bioethanol production. Of all
conventional crops grown in Europe, sugar beet yields the highest amount of
bioethanol per hectare (5.6 t or 7,100 litres) [144]. Several factories for the
production of ethanol from beet are operational or under construction
(www.ebio.org). For example, Südzucker presently has three production facilities
installed, and is rapidly expanding its production capacity to 1 million cubic meters
per year. The conversion of existing sugar processing facilities is also an option
[225].
There is a technical issue of relevance to future breeding programmes of sugar beet
designed specifically for biofuels, biopolymers or chemicals rather than crystal
sugar production. Current varieties have been optimised for juice purity � since the
presence of such as potassium, sodium, and α-amino-N (amino acids, betaine and
other nitrogenous compounds) affects the crystallisation process from the aqueous
beet extract and increase the amount of sucrose lost to molasses [114, 115].
Besides sucrose concentration, this is the main factor determining the relative
suitability of sugar beets for processing or �technical quality�. During processing,
glutamine and asparagine decompose to pyrrolidone, carbonic acid and ammonia.
These compounds decrease the buffering capacity of the factory juices and
increase the need for lime. Reducing sugars such as glucose and fructose also
decrease the alkalinity. Moreover, reducing sugars react with nitrogenous
compounds according to the Maillard reaction, staining the sucrose crystals and
32
diminishing white sugar quality [135]. Currently, breeders use the respective
technical quality as a major selection criterion for developing new varieties.
Therefore, during the past decades the composition of cultivated beet has largely
improved with regard to the technical quality [165].
If the entire beet crop is processed as a whole for biofuels then purity is not relevant
and breeding programmes will need to alter direction to other traits of relevance for
industrial production of biofuels. The traditional breeding objectives of high sugar
and low impurities content can be replaced by simple selection for high levels of
fermentable sugars [32, 33]. It has been suggested that a 10% yield increase is
easily accomplished. Such higher yielding varieties have been found many times in
breeding programs, but were discarded due to poor juice quality making them of no
interest to the conventional sugar industry. Studies conducted in the 1980s
concluded that fodder beet cultivars would constitute the most promising starting
point for the development of a fuel or energy beet [247].
An alternative is that only the side streams of beet tops, sugar beet pulp and
molasses are used as fermentation feedstocks for biofuels or for other purposes �
this can be considered as a more conventional option, but does not aid the
emerging issues of sugar reform (Table 3, strategy 1). In the context of producing
novel chemicals in a sugar beet platform, their presence could affect technical
quality if the conventional route were pursued. As a designer industrial crop in which
the entire beet was fermented, this would not be a problem. However, the chemical
would have to be recovered prior to fermentation (Table 3, strategy 3).
33
Table 3 Strategies for the production of feedstocks in sugar beet
Strategy 1 Strategy 2 Strategy 3 Sugar production,
and novel chemicals from pulp and/or molasses
Conversion of whole beet to bioethanol / biobutanol
Production of novel chemicals, conversion of remaining biomass to biofuel
Based on existing processing
Strong political and financial support
Non-food
New applications of profitable crop
No GM required New applications of profitable crop
Advantages
Established technology
Combination of GM and food product
Depends on subsidies
GM required
Limited to products that do not interfere with technical quality
CO2 and energy balances not great
New processing technology required
Disadvantages
Does not aid CAP-reform
Competition with white biotechnology
Prediction /forecast
Uptake hinges on acceptance of GM-beet for food
Rapid uptake already in progress (71% increase in 2006)
Uptake hinges on acceptance of GM-beet for non-food
3.2.5 Novel co-products and processing technology
There have been a number of suggestions for altering sugar beet to improve its
potential as an industrial crop platform [139], and the prospects of making novel
products, technical polymers and chemicals in plants has been discussed [13, 105,
139, 241]. These are outlined and discussed in section 3.11.
One case study has been published in scientific literature. Transgenic beets
producing fructans (a low caloric sweetener) have been generated by the
expression of synthases targeted to the vacuoles where the sucrose accumulates.
The fructosyltransferase of Helianthus tuberosus expressed in the transgenic beet
did in fact convert sucrose into low-molecular-weight fructans. An astonishing 40%
34
of the taproot dry weight was found to be fructans. Moreover, this was
accomplished without detriment to the host plant [228]. Given the change in sugar
quantity/quality, the beets would no longer be used in the traditional processing
facilities: processing for fructan production would have to be included in a
processing scheme for bioethanol production in which the fructans would be
extracted prior to fermentation.
In the context of current processing practices, it is conceivable that novel side-
streams of new products could emerge from the existing processing flow chart of
operations (Table 3, strategy 1). Soluble chemicals will end up in the molasses from
which they may be recovered e.g. by technologies building on chromatographic
molasses desugaring (MDC). Insoluble chemicals or polymers are likely to become
part of the pulp fraction, and may be extracted using solvents.
However, new chemicals could interfere considerably with technical quality and
sugar crystallisation in the standard operations. As discussed in earlier sections,
use of the entire beet for industrial production of biofuels and other bioproducts
would bypass the problems associated with sugar for human food production. It has
also been suggested by the industry that even if co-products were present, an
easily accessible sugar fraction (perhaps only 20-30% of total) could be recovered
at low cost and the sugar-rich residue (70-80% of total) used for fermentation.
Alternatively, the ratio between sugar and ethanol may be optimized according to
the actual commodity prices. In this scenario, processing technology for co-products
would have to be designed tailor-made for the particular chemical or polymer.
Amongst all herbaceous crops, sugar beet may be the most thoroughly studied crop
species and therefore it can be expected that technical problems that arise in the
future could be overcome easily. Although there appears to be very little visible
enthusiasm by the sugar industry for innovation [259], in part due to the perceived
risk by food companies of becoming associated by the public with GM technology
and transgenic plants, innovative and profitable uses of beet are clearly in the
interest of farmers and the processing industry.
35
3.3 Genetics
Turning sugar beet into a bioproduction crop requires knowledge on fundamental
aspects of sugar beet biology, including genetics. It also requires the availability of
tools for breeding and genetic transformation. Taxonomy helps to judge safety and
actions to prevent gene flow and outcrossing.
3.3.1 Taxonomy
Modern sugar beet is derived from germplasm of the White Silesian Beet, a fodder
beet (Beta vulgaris var. rapacea). Central European beets are presumed to be
descended from those used in Arabian horticulture in Spain. These plants were
taken to the Netherlands, where they were cultivated beginning in 1500, and then to
the Palatinate region, later spreading throughout Germany as �Burgundy beet�.
During the sixteenth and seventeenth centuries, red and yellow beets became
increasingly common as salad vegetables. Fodder beet cultivation only began to
increase in the course of the eighteenth century. The crop was introduced into the
USA in 1800, where it became known as garden beet. Sugar beet was introduced to
North America around 1830 and to South America circa 1850 [163].
Beet belongs to the genus Beta, the family Chenopodiaceae, the section Vulgare,
and the species Beta vulgaris. The section Vulgare encompasses the wild beet
species B. maritima, B. macrocarpa, B. patula and B. atriplicifolia [256], all of which
are cross compatible [237]. Beta vulgaris comprises several cultivated forms of B.
vulgaris subsp. vulgaris. Cultivars include leaf beet (var. cicla) and root beet (var.
esculenta). Recent molecular data have confirmed the old idea that the wild sea
beet (Beta vulgaris ssp. maritima) is the progenitor of all domesticated beet [11,
194]. This wild form is a �beach plant�, thus salt-tolerant. It is native to the
Mediterranean and Atlantic coasts of Europe (Figure 4). The cultivated sugar beet is
a biannual, which grows vegetatively in its first year and develops a large fleshy
taproot that contains the food reserve for the second year of growth. In this stage it
36
is harvested. If allowed to overwinter it becomes reproductive, and it forms a stem
terminating in an inflorescence.
Beta sect. Beta encompasses closely related wild, weedy, and cultivated forms of
which more than 4350 unique accessions are maintained in seed collections. Since
Beta germplasm is held by various genebanks in the world an internationally
accepted classification system should exist, capable to transmit reliable information
on Beta genetic resources. A fully consolidated taxonomy of the genus and a
consistent classification system for Beta sect. Beta is not available. However, the
accurate classification of Beta accessions would be a fundamental prerequisite for a
purposeful choice of germplasm from collections (see Chapter 1 of [32] for an
overview of taxonomic issues, management and safeguarding of germplasm).
Figure 4 Beet cultivation. Triangles show the geographical distribution of
sugar beet cultivation.
37
3.3.2 Genetics
Beta vulgaris is a diploid plant species with 18 chromosomes and an estimated
genome size of about 758 Mb per haploid genome, 60% of which consists of highly
repetitive DNA sequences. Most of the sugar beet grown since 1960s has been
triploid, because these hybrids displayed a stronger disease resistance, and yielded
up to 10% more than the parental average yield. Triploids are produced by crossing
tetraploid parents with diploid male sterile plants. They are usually doubly sterile
because of chromosome imbalance and cytoplasmically inherited male sterility.
Diploid varieties (2n=18) are now used more frequently, as they allow the
production of true F1 and 3- and 4-way crosses in breeding programs. Certain
inbred sugar beet lines are reported to have developed apomixis and are thus able
to reproduce without fertilisation [45, 87, 97, 128, 208]. A summary of breeding
systems and seed yield of Beta vulgaris and relatives is given by [95].
3.3.3 Tools
Industry, academic labs, and dedicated sugar beet research institutes (no less than
22 in Europe) make great progress in breeding and the molecular genetics of sugar
beet. A thorough overview of the impact of molecular biology and biotechnology on
sugar beet breeding and development is given in Chapter 4 of [32]. The main result
is a rapidly increasing understanding of genes, genomes, and in extenso also
cellular biochemistry and physiology. In the long run, this knowledge will
dramatically increase the efficiency of plant breeding for traditional sugar producing
varieties, but significantly in the context of an industrial crop platform, also for new
varieties targeted at new products.
Sugar beet has become a model organism to study the organization of nuclear
genome due to its relatively small genome size and the fact that FISH analysis can
accurately locate sequences within interphase nuclei and the metaphase
chromosomes. Linkage maps are available [125].
38
Herwig et al. have applied oligonucleotide fingerprinting (ofp) to close to 160,000
sugar beet cDNA clones, which revealed 30,444 ofp clusters likely to represent
different genes. Sequencing of 11,000 clones confirmed that 89% of ESTs did
represent different genes, which indicates that the 30,444 ofp clusters represent up
to 25,000 genes [112]. A BAC-library, with an average insert size of 120
kilobasepairs, and assumed 6-fold genome coverage is also available [169]. The
mitochondrial genome of sugar beet has been sequenced [138]. Within the
framework of the German genome project GABI four projects deal with sugar beet.
GABI-BEET provides tools, methods, information and genetic materials for the three
other more breeding oriented projects GABI-BOLT, GABI-SWEET, and BREATH-
LESS GABI. The main goals of GABI-BEET are 1) EST sequencing and related
bioinformatics; 2) development of high density SNP based marker maps; 3)
genomic studies of cultivated and wild Beta species; and 4) construction of large
insert libraries. There are currently no plans to sequence the genome.
Sugar beet transformation is an established method and has been used to introduce
1) herbicide resistance genes [164]; 2) coat protein encoding genes of the
rhizomania-causing virus BNYVV [143]; 3) and a single gene from Jerusalem
artichoke encoding an enzyme that converts more than 90% of the sucrose in the
transgenic beet to low molecular weight fructan [228]. Transformation of sugar beet
plastids has not yet been successful yet, and is one of the objectives of a STREP-
project named Transcontainer (http://www.transcontainer.wur.nl/).
3.4 Breeding
3.4.1 Historical overview and background
Sugar beet is unique in that it started to be developed at a time when modern
genetic principles were becoming understood in the late 1700s. Therefore, the
genetic base of sugar beet is thought to be narrower than for many open-pollinated
crops. Initial goals of breeding were improved concentration and extractability of
sucrose. Host-plant resistance to insect, nematode, and disease pests were
39
neglected, but as production areas expanded, pests sometimes severely limited
production. Thus, resistance to pests has become an important target. The first
systematic attempts to screen exotic and wild beet germplasm for disease
resistance were begun at the beginning of the 20th Century. Over 3,000 evaluations
described levels of resistance of sugar beet and wild beet accessions to 10 major
disease and insect pests of sugar beet. There is a lag time in sugar beet of 6 to 10
years between starting a germplasm development program and releasing the first
developed germplasm.
An important development is the change to monogerm seed. This obviated the need
for thinning, but more effective plant protection (i.e. more pesticides and herbicides)
was required. The monogerm trait had to be introduced in part of the diploid
multigerm gene pool. This led to subsequent search for sterility maintainer
genotypes and development of their genetic male sterile counterparts. Therefore,
strict inbreeding and back-crossing became the major selection methods. Many
open-pollinated diploid multigerm populations were lost because they were not of
immediate value in this process [32].
3.4.2 Conventional and marker assisted breeding
As an established and valuable crop, an extensive literature exists on conventional
breeding targets and methods. Comprehensive overviews can be found in the
monographs: Genetics and Breeding of Sugar Beet (2005), edited by Bianchardi et
al. [32]; and Sugar Beet (2005, World Agriculture Series), edited by Draycott [77].
In general, breeding serves to increase the sugar yield per hectare and year, while
minimizing input of labour, fertilizer, herbicides, pesticides, water. The major focus
of this effort thus was technical quality, i.e. optimising the amount of sugar that can
be crystallized in the processing facility [165]. This has resulted in annual sugar
yields of 11 t ha-1, with a labour requirement of 20-50 man-hours ha-1 [77]. Sugar
yields per ha and year increase on average by 80 kg per year [166].
40
Breeding sugar beet for biofuels production has considerable impact on the
breeding objectives, as technical quality is no longer an issue. In combination with
new methods such as metabolic profiling, significant yield increases can be
expected [176]. Further new targets are the development of a hardy winter beet that
can be sown in autumn. This beet would profit from more growing days, leading to
increased yields. It could also be harvested earlier, thus extending the processing
campaign. A beet that could be stored longer would also extend the processing. It
can be expected that beet used for bio-ethanol and other products can be stored
much longer without affecting the yield too much, because the conversion of
sucrose to polymers or other compounds is only problematic for sucrose production.
Both in Europe and the United States, sugar beet variety improvement and seed
production are carried out primarily by private companies. However, the USDA
developed most of the varieties grown in the first half of the 20th century in the
United States and current variety development often uses genetic lines derived from
these varieties.
Marker-assisted selection (MAS) is already used in practical breeding work, but is
limited to resistance genes for beet cyst nematode and rhizomania. The use of MAS
for quantitative traits is less straightforward because multiple interacting genes are
involved, and the translation of present knowledge in effective MAS strategies for a
trait like sugar or biomass yield is likely to be quite an undertaking [38].
3.5 Susceptibility to abiotic stresses
Sugar beet is grown in a wide range of environments and climatic conditions, from
tropical countries to Southern Scandinavia, and is derived from a species that grows
under a wide variety of challenging conditions from beaches and rocky cliffs
exposed to sea spray, to sites with shallow, sandy soil. Thus, beet shows a
remarkable tolerance to both salinity and drought [77]. Drought tolerance
mechanisms often involve accumulation of osmotically active solutes and indeed
transgenic sugar beet accumulating fructans to low levels (max. 0.5 % of dry
weight) showed significantly better growth (+25-35 %) under drought stress than
41
untransformed beet, whereas under well-watered conditions, no significant
differences were observed [201].
Despite the drought tolerance of beet, lack of water is a major limitation to sugar
beet yield in many geographical regions that do not use irrigation. The impact of
future climate change on sugar beet yields has been assessed over western Europe
using future (2021�2050) climate scenario data and the Broom�s Barn simulation
model of rain-fed crop growth and yield [131]. The results emphasise the
importance of crop breeding for drought tolerance (see section 2.9.1).
3.6 Susceptibility to biotic stresses
As an established crop, a great deal of information is available on viral and bacterial
diseases, weeds, and pests of sugar beet. These are general issues in sugar beet
cultivation and are not directly relevant to the production of polymers or chemicals.
Comprehensive overviews can be found in the monographs Sugar Beet, edited by
Philipp Draycott [77], Genetics and Breeding of Sugar Beet (2005), edited by
Bianchardi et al. [32], and the Zuckerrübenkompendium (2006) by Manfred Bartels
[25]. Young sugar beet plants are vulnerable amongst others to the diseases
Rhizoctonia, Cercospora leafspot (caused by a fungus), Aphenmyces and Pythium,
and various insects. Older plants suffer amongst others from curly top (a viral
disease carried by the beet leafhopper) and Rhizomania (a viral disease carried by
a soil fungus). Nematodes and beet weevils also cause serious problems
depending on the growth region.
3.7 Agronomy
As for all established crops, a great body of information is available on agricultural
practices in sugar beet cultivation. Comprehensive overviews of the agronomics of
sugar and fodder beet can be found in number of monographs including for
example, Sugar Beet, edited by Philipp Draycott [77], Genetics and Breeding of
42
Sugar Beet (2005), edited by Bianchardi et al. [32], and the
Zuckerrübenkompendium (2006) by Manfred Bartels [25].
In Europe, typical soils used for sugar beet are loamy sands, loams, silty loams or
peaty loams, mostly in flat areas. The yield of sugar beet depends primarily on site
and year effects, whereas the influence of agronomic practices is much lower.
Taproot yield is correlated with thermal time and the amount of water available in
the soil.
Sugar beet is grown in 23 of 27 member states of the EU [48] (faostat.fao.org), and
covers 2% of the usable agricultural land. It is a rotation crop planted in systems
comprised of mainly cereals but also potatoes, maize, and protein and oil producing
plants. In the UK and Sweden beet cannot be grown more frequently than every
third year. In France, two beet crops are typically followed by at least 2 to more than
4 years with another crop. In cooler areas, it is treated as an annual crop sown in
spring and harvested in autumn, while in hot areas it is planted in fall and harvested
in the next summer.
In 2005 sugar beet was grown on 2.2 million hectares, with an average yield of 57.4
t ha-1 (beet wet weight), a total production of 126 million t, and a typical sugar
content of 16-18%. Sugar yields vary from about 6.5 t ha−1 in Finland to more than
15 t ha−1 near the Black Sea, (Ukraine, Krasnodar), where it is warmer and sunnier,
and where the growing season is longer [200]. Simulated stress-free yields also
increase from the West, e.g. Ireland with 8 t ha−1 (where the weather is cool in early
summer and where it is often cloudy) towards the East, e.g. Poland with more than
11 t ha−1, as the climate becomes more continental and solar radiation levels
increases. Several regions with sufficient rain show high yields, which are also
favoured by low annual variability. This is the best combination for a secure, highly
productive industry. Northern France, large areas of north and central Ukraine, west
and central Poland (on the better soils) and southern Germany are prominent in this
category. Drought losses are greatest (over 40%) in the eastern Ukraine and
43
southern Russia [200]. A theoretical yield for Germany and comparable agroclimatic
regions of 24 t ha-1 (dry weight) was calculated [136].
Using a computer model, Jones et al. (2003) assessed how sugar beet yields may
change in the future as a result of climate change. They predicted that overall sugar
beet yields are expected to rise during the period 2020�2050 by around 10% due to
increasing atmospheric CO2 concentration [70, 131]. Northern European regions
show an additional increase in yield (due to warmer springs), which is more than
offset in western and central Europe by increased losses due to drought stress, with
weighted average annual yield losses rising to 18%. Year-to-year variability in yield
will also increase (50% higher coefficient of variation); this has important financial
implications for the industry. The results stress the importance of breeding for
drought tolerance [131].
3.8 Environmental impacts
3.8.1 Agronomical impacts and water requirement
Sugar beet has a high absolute water demand of 300 to over 500 mm. In northern
and central Europe, less than 10% of the total sugar beet area is irrigated, whereas
most sugar beet grown in southern Europe must be irrigated to maintain
productivity. Water use efficiencies for sugar beet dry matter production (WUEdm)
sown in spring in Northern Europe and America are reported to be between 4.6 and
5.6 g kg-1 of used water [43].
Water Use Efficiency (WUEdm) values ranged from 2.1 to 10.0 g kg-1 in several
environments [79]; in experimental areas with seasonal water use close to Southern
Italy conditions (600�900 mm), the values ranged from 2.3 to 5.8 g kg-1, while in
Sweden values around 8 to 10 g kg-1 were obtained [217]. Water use efficiency for
sucrose production showed a linear increase with water irrigation amount, ranging
from 0.7-1.6 g kg-1. In Southern Italy WUEdm of 2.83 g kg-1 were obtained for both
beets sown in spring and autumn [211].
44
Beet fields are vulnerable to wind erosion as well as water erosion, since they are
often left bare over winter [48]. One way to prevent this is separating fields by a
single-row shelterbelt of coppiced trees. Soil compaction by heavy equipment is
also an important issue in sugar beet cultivation. The WWF claims that the loss of
soil during harvesting (tare: soil and other materials sticking to the beets) can reach
10-30 percent of the total beet harvest weight [276]. According to Asadi this number
varies from below 2% to over 8% [17]. Usually, tare is transported back to the fields.
The requirement for N-fertilisation for sugar beet depends on the N content of the
soil. Since there is a strong negative correlation between nitrogen and sugar
content, excessive application of nitrogen is rare as it reduces the value of the crop.
The average beet crop requires 200 � 250 kg N ha-1. On most soils about 100 � 150
kg N ha-1 is mineralized from leafy intercrops such as Lucerne worked into the soil,
resulting in an effective fertiliser demand of around 100 N ha-1 [165]. Based on the
simulation model of Wageningen University the nitrogen input for sugar beet is 290
kg and 3.8 kg of active ingredients for pesticides are used in the Netherlands [107].
In field trials in the Netherlands, N uptake of beets was 275 kg N ha-1 year-1 [272].
Remaining sugar beet tops on the field after harvest may increase N leaching in
winter, but also increases N uptake and yield in succeeding crops [248].
Sugar beet is very vulnerable to weed competition, sometimes leading to a
complete loss of the crop. This is due to the low height of the crop and the late
canopy closure. Thus, weeds should be controlled nearly completely, at least until
the eight-leaf stage. Late germinating weeds may also constitute a problem,
especially if they�re tall and spreading. These can overgrow the canopy, and
suppress the crop significantly by shading. Finally, weeds can cause serious
technical problems during harvest [164]. As a consequence, extensive herbicide
treatments are required, although these tend to have acceptable ecotoxicological
behaviour, and contamination of water and soil is insignificant. The use of plant
protection products has halved since the 1980s and is now at approximately 4 kg
ha-1 y-1. This was accomplished by more rational plant protection schemes and the
inclusion of insecticides in pelleted seed.
45
Beet grown for the production of bioethanol may change many parameters. For
example, it is no longer crucial to have strict control of nitrogen to maintain technical
quality. On the contrary, more intense nitrogen fertilisation may increase beet yield
by 5 % [264].
3.8.2 CO2 emission and carbon sequestration
According to Tzilivakis, energy inputs of sugar beet production in the UK ranged
from 15.72 to 25.94 GJ ha-1. The large spread in these values was mainly
determined by crop fertilisers, which in turn were determined by the soil type. Other
important factors were tillage, input of livestock manure, irrigation, and � in the case
of organic farming � the distance to the sole processing factory for such beets in the
UK. Crop protection energy costs were not critical.
The amount of energy in dry matter was between 7.3 and 15 times higher than the
amount of energy invested (between 195 and 234 GJ for conventional, and 132 GJ
for organic sugar beet). This was clearly better than that of other conventional crops
such as winter wheat, where the yield is 109 GJ. Thus, from an efficiency point of
view (also from the land-use perspective), sugar beet is well suited as raw material
for producing bio-ethanol and other renewable products. In a comparable long-term
study based on German agriculture, similar results were obtained, though with a
larger spread [123].
Beet ethanol
Considering the use of beet to produce fuel, the energy efficiency (released energy /
non-renewable energy used) of beet ethanol is 2.05 (compared to 0.873 for
gasoline). Prospective scenarios indicate that foreseeable technical progress in the
entire production chain may give improvements of more than 140% in the energy
performance of the sugar beet chain per surface unit [48].
A study by the USDA Foreign Agricultural Service [94] shows that ethanol
production from beet compares favourably with wheat with regard to fuel efficiency
46
and leads to a significant reduction in gas-house gas emissions (Table 4). A similar
study by ADEME/DIREM concludes that that the energy efficiency ratios (released
energy divided by non-renewable used energy) of wheat and sugar beet ethanol
chains are 2:1, in comparison with the value of the gasoline chain, which is 0.87:1.
For both crops, the agricultural stage contributes only 20% to the energy balance
[1].
Table 4 Estimates from European Studies on Ethanol Production Efficiency
Average ethanol production efficiency
Fuel process energy efficiency
Well-to-wheels GHG emissions compared to gasoline
Wheat-to-ethanol (Based on 5 studies)
356 litres per ton of feedstock
0.91 Input: 136.5 e.u. Output: 150.0 e.u.
Between 19% and 47% reduction (Average 32%)
Sugar beet-to-ethanol (4 studies)
86 litres per ton of feedstock
0.67 Input: 100.5 e.u. Output: 150.0 e.u.
Between 35% and 56% reduction (Average 46%)
Source: International Energy Agency; e.u. = energy units; GHG = greenhouse gas
Carbon sequestration Quantitative data on carbon sequestration in sugar beet cultivation are rare, also because sugar beet is grown in rotation with other crops. The overall crop sequence must be compared, which is not trivial as this differs from region to region, and with time. One study makes a detailed analysis of different scenario�s to grow sugar beet in the UK. On a per ha basis, a yield of 52 t beet wet weight (16% recoverable sugar), a net margin of £ 560, consumption of 21.4 GJ of energy, emission of 1.4 equiv t of CO2, 3.3 kg nitrogen leached, 15.2 kg nitrogen lost to denitrification and a pesticide ecotoxicity of 26 (which is low compared to potato at 230, and winter wheat at 35) [253]. The scenarios are based on the assumption that beet tops are left in the field, which is common practice [254]. Using the whole beet, as is a possible scenario in using beet for biofuel production, would negatively impact soil quality.
47
Current cultivation practices that include tillage lead to loss of soil organic carbon. The level of carbon sequestration and soil organic matter depends strongly on the cropping system applied. Tillage in beet cultivation systems has been found to promote organic matter breakdown leading to declines in soil structure and health. Reduced tillage coupled with annual cropping has been proposed to minimize or halt the loss of soil organic carbon content and maintain soil productivity [263]. The effects of improved cultivation practices are only visible on a long term. Ecological processes are slow and the determination of the amount of carbon sequestration takes many years. 3.8.3 Gene flow and biosafety
Gene flow and biosafety are major issues in the widespread cultivation of transgenic sugar beet and these issues will necessarily impact on the design of new beet varieties as an industrial crop platform for novel biopolymers and chemicals. The potential for gene flow is given by the fact that sugar beet belongs to the same species as fodder beet, Swiss chard, red beet and the wild sea beet, which grows along the coast. It cross-hybridises with other closely related Beta species. Cultivated, wild and weed forms are often difficult to distinguish [96], also sea beet genetic resources have been extensively used in conventional breeding programs [27]. Moreover, beets are wind-pollinated and out-crossing through self-incompatibility. Seeds (especially of sea beet) can be dispersed by water, allowing gene flow over long distances [60]. The possible gene-flow routes are summarized in Figure 5. Flowering weed beets in sugar production areas have rapidly emerged as a serious
problem since the early 1970s in Europe. This weed beet appears to be
phenotypically different from volunteer sugar beet in that it produces more seed and
usually does not need a vernalization [108]. Sugar beet can also become a serious
weed problem through remaining roots/crowns left in the field post-harvest.
Currently, this problem is successfully prevented by eradication with effective
herbicides.
48
wild (ruderal beets
sugar beet seed production field
pollinators 4N or 2N
seedbearers
surroundings of the seedproduction field
South-west France Northern France
North Sea and Channel coast
wild sea beets
crop-wildhybrids
variety 3N or 2N(with some bolters)
sugar beet field 1
sugar beet field 2
weedbeets
weedbeets
pollen flowseed flow
Figure 5 A schematic presentation of the possibilities of gene flow by seeds and pollen in the sowing seed-production area (left) and in the sugar-
production area (right) [71]. The seed bearers are male-sterile, the pollinator plants are hermaphrodite; all other plants can be both. The pollinator plants can be tetraploid (4N) or diploid (2N), leading to triploid (3N) or diploid (2N)
varieties, respectively; all other plants are usually diploid. Gene flow is possible between all forms present in the field (weed beets, transgenic plants, diploid F1 crop–wild hybrids and triploid variety bolters). The appearance of
transgenic weed beets is possible (e.g. hybrids containing both a bolting allele and a transgene), which can best be retarded if the transgene for
herbicide tolerance is incorporated into the tetraploid pollinator breeding line.
49
When beets are grown for seed production, it is imperative to prevent pollination by
other beet variants, weed beet or any wild relatives such as sea beet, which are
difficult to distinguish. In the case of transgenic sugar beet, transgenes may be able
to persist in weed beets derived from bolters or volunteers. Although pollen usually
represents a significant vector for the spread of genetically modified traits, seed flow
may also have a strong impact in connecting wild and crop relatives within the
complex Beta. The longevity of seed is often underestimated: under laboratory
conditions 50% of sugar beet still germinate after 8 years [16].
Hybridisation between transgenic plants and any other B. vulgaris variants may be
difficult to prevent. Introgression of these plants and cultivation under selection (for
example for a herbicide resistance) could produce annual weed beet containing the
transgene. Thus, escape of transgenes to crop weeds must remain a realistic
scenario for sugar and fodder beet.
Mitigation of gene flow
Field trials with transgenic sugar beet show that competition strongly influences
gene flow. It is stated that ecological implications due to the introduction and spread
of BNYVV virus-resistant transgenic hybrids will be observed only in those feral
Swiss chard and wild beet populations where fitness is significantly influenced by
high infestations of BNYVV [27]. Although the hybridization rate of transgenic plants
and wild-types does not show significant differences, the bolting rate of the
transgenic traits is significantly lower, probably due to pleiotropic effects. The
transgenes cause a lower competitiveness as indicated by the term �cost of
resistance� [27]. The observed phenotype demonstrates that genetic engineering
may cause unexpected effects, which would probably reduce the risk of gene flow
to wild relatives of cultivated plants.
As this will not generally prevent gene flow, reliable measures to reduce the risk of
transgene escape [28] are required. These include the use of doubly sterile triploid
varieties, varieties with a decreased frequency of bolting, and the production of
50
seed only in a highly regulated manner and restricted to areas distant from the
coast in southern Europe (to avoid transfer of the transgene to sea beet).
An interesting option is asexual reproduction or apomixis. Apomixis is the process
of asexual reproduction through seed, in the absence of meiosis and fertilization,
generating clonal progeny of maternal origin. Certain inbred sugar beet lines are
reported to have developed apomixis and are thus able to reproduce without
fertilisation [45, 87, 97, 116, 128, 208]. Apomixis has a number of general
advantages in agriculture, which include fixation of hybrid vigour in crop plants;
survival and immediate fixation of combined genetic resources, including wide-cross
progeny; and more rapid breeding programs. Attempts are ongoing to identify genes
from Beta corolliflora involved in apomixis [87]. If successful, the relevant genetic
elements could be transferred to common sugar beet lines.
Geneflow can also be mitigated by Cytoplasmic Male Sterility (CMS). CMS is
already used to maintain the monogerm phenotype in modern sugar beet lines.
Male sterility is under the control of the cytoplasm with nuclear genes restoring male
fertility, although cultivated and wild beet may have different nuclear and
cytoplasmic components [39, 40, 193]. They can hybridise freely and hybrids are
spontaneously formed in the wild and in seed-production fields.
Safety of plant-produced industrial products
An extensive report by the Office for Technology Assessment at the German
Parliament concludes that due to the early stage of GM-crops producing industrial
compounds, there is no advanced risk-discussion in progress. However, many new
plant products (especially the biopharmaceuticals, but also chemicals and
biopolymers) could be physiologically active, thus pose inherent health risks [219].
The presence of anti-nutrients or toxic compounds due to breeding or genetic
engineering is in fact a general issue in the development of new crops. In the case
of beet, specific rules have been drawn up to help assess the safety of new variants
[10]. If beets are developed for the production of chemicals and biopolymers, their
51
safety will have to be assessed as well [170]. Mitigation of risks includes those
mentioned in section 3.8.3. It is clear that potentially noxious or toxic substances
must be prevented from doing harm to consumers or wildlife. Common scenarios for
concern are problems arising from commingling as a result of errors during seed
production, seed drop during harvest, transport and processing, and mix-ups of
harvested materials.
Whilst food from GM-crops is likely to remain controversial in Europe, industrial
uses of GM-crops may be more likely to be accepted, provided that cultivation and
processing is strictly separated from food crops. In the case of beet, processing
facilities for sugar refining and for biofuel production are completely distinct,
involving different capital infrastructures and requiring the construction of new
industrial plants for conversion to bioethanol / biobutanol. It is probable that each
distinct processing plant / use will require a cultivation area as close as possible to
the processing site. It is therefore possible to envisage completely separate
cultivation areas for the different uses of the beet.
3.9 Economics 3.9.1 Cultivation costs and net margins In assessing the economic potential of sugar beet as a feedstock platform for this
sector of the bioeconomy we have looked at data for Italy, Germany, Poland,
Sweden and the UK. Each of these countries represents a different climatic region
of the EU with potential for the successful cultivation of different crops. It is not our
intention to propose monocultures in any of these regions but to draw out significant
differences that currently exist and provide a basis on which future cropping plans
can be considered. In addition, it will also be necessary, in the longer term, to
consider the impact of climate change on agricultural production. In the case of
sugar beet, modelling of the effects of climate change in Europe has been
performed [70, 131] (see section 3.7 for more detail).
52
Table 5 summarizes the generated data. It sets out the value per tonne of beet yield
and the value of the per hectare beet yield. The sugar beet revenue is based on the
minimum guaranteed beet price in 2007/2008 of � 27.4. No value has been given to
the beet tops and leaves produced as a by-product of the crop. The value of the
single farm payment has been shown in the table and net margins calculated to
demonstrate the effect of including and excluding this subsidy.
It should be noted that these calculations are based on general assumptions and
that the production costs for individual farmers will be different. Also, the net
margins have been calculated using �assignable� fixed costs, to achieve a
comparison of the relative profitability of the different systems. Therefore, because
the full fixed costs of the farm are not included, the net margins do not represent the
actual profit from each enterprise.
Full details of the data used and assumptions made in the preparation of the
economics information for this report are available on the EPOBIO website
www.epobio.net and the following sources: [2, 18, 84-86, 88, 190, 244, 245]). The
variable costs shown in the tables include: the cost of seed/planting material;
agrochemical inputs � fertiliser, pesticides and herbicides; and variable costs of
straw baling, primarily identified in literature as baling string. Fixed costs include:
cost of machinery for cultivation, drilling and the application of agrochemicals;
combining, including the management/handling of straw; labour costs; land costs;
and the on-farm costs of collection, drying and storage.
The economic calculations based on these data and assumption indicate a net loss
in Italy (mainly due to the low yield in Italy) to net margins in the other four countries
ranging from � 515 in Germany to � 775 in Sweden. Without subsidies, a much
greater loss is made in Italy, while profits in Germany decrease to � 198. These net
margins are much lower than obtained before the CAP-reform, when typical net
margins were over � 1000 per ha. In the Netherlands in 2005, for example, sugar
beet yielded � 1350 per ha [9].
Table 5 Calculation of net profits for sugar beet grown in five European countries
Unit UK Poland Germany Italy SwedenYield t ha-1 56.9 42.7 57.8 43.6 51
� t-1 yield 29.97 27.07 27.38 27.38 32.64 Crop revenue � ha-1 1705.29 1155.85 1582.56 1193.77 1664.39 Single farm payment � ha-1 319.64 93.44 316.55 554.23 242.32
� t-1 yield 35.59 29.26 32.86 40.09 37.39 Total revenue (crop and single farm payment) � ha-1 2024.93 1249.29 1899.11 1747.99 1906.70 � t-1 yield 10.40 7.90 7.67 17.11 7.11 Summary of variable costs � ha-1 591.91 337.37 443.53 745.80 362.79 � t-1 yield 14.45 8.47 16.28 23.59 15.08 Summary of fixed costs � ha-1 822.48 361.67 940.99 1028.57 768.92 � t-1 yield 24.86 16.37 23.95 40.70 22.19 Total costs
� ha-1 1414.39 699.04 1384.52 1774.36 1131.71 � t-1 yield 5.11 10.70 3.43 -13.32 10.44Net margins (without single farm payment) � ha-1 290.90 456.81 198.05 -580.60 532.67� t-1 yield 10.73 12.89 8.90 -0.60 15.20Net margin (with single farm payment) � ha-1 610.54 550.25 514.59 -26.37 774.99
54
Because of the much lower net margins, sugar beet cultivation in the EU has
decreased in 2005 and 2006. However, because of the locked supply chain, beet
processors will pay farmers more for beet than for winter wheat, which yields about
� 600 in the Netherlands [137], thus guaranteeing an adequate supply for the
processing campaign.
Because net margin of sugar beet cultivation has decreased substantially due to the
CAP-reform, other uses of beet are being considered. These include the production
of bioethanol [247], but on a longer time frame also the production of chemicals for
added value [137, 139].
3.9.2 Bioethanol
Although the production cost of beet ethanol without subsidies is approximately
three times as high as that of cane ethanol [111], political support makes the
production in Europe of ethanol from beet a viable alternative to sugar production.
The ambitious targets set by the European Union for biofuels (share of total use
5.75 % in 2010) is leading to the implementation of policies such as tax reductions,
incentives for research and development, mandatory blending requirements, and
investment subsidies. The EU supports biofuel production �to promote sustainable
farming, protect the countryside, create additional value added and employment in
rural areas, reduce the cost of farm support policies, and diversify its energy
supplies.� Since July 2006, sugar beet production qualifies for set-aside payments
when grown as a non-food crop as well as for the energy crop aid of � 45 euro per
hectare. Sugar for bioethanol is excluded from production quota. In addition, food
security issues and the destruction of rainforest due to large-scale production of
biofuels in third world countries could lead to barriers on imports [111]. Koops et al
have calculated that the net margin from energy production using all biomass is in
the range of � 600 per ha, which is very similar to that obtained for beet used in
sugar refining [137].
55
In 2004, 0.8% of sugar beets went towards bioethanol [94], and the total EU
production of bioethanol (from wheat and beet) was just over 490 thousand tonnes.
Major beet sugar producers such as Südzucker, Tereos, British Sugar, Cristal
Union, and Nedalco (Table 2) now operate beet ethanol plants (www.ebio.org),
while others (Nordzucker, Cosun) are building or planning such facilities. In 2006
ethanol production capacity from beet increased by 71%. The production cost of
beet ethanol is largely determined by biomass feedstock prices, which can account
for 55 - 80% of the final price of ethanol. As the conversion of sugars to ethanol is a
mature technology, technological improvements that significantly reduce the current
production costs are not likely.
Two production cost analyses, published by the Biomass Technology Group (Table
6) and the International Energy Agency (Table 7), relate to the economics of ethanol
production from wheat and beet. The results are based on feedstock prices reported
by F.O. Licht�s (April 2006) of � 24.10 t-1 or in the BTG study � 26.2 t-1. The IEA and
BTG studies give a 7 or 3 cent credit for by-products, respectively. For a detailed
discussion and comparison of these datasets see [94]. Other studies cite production
costs of bioethanol from beet of � 0.32-0.53 (ECN-study cited on www.eubia.org) or
� 0.59 per litre [81].
Table 6 Bioethanol production costs from beet in the EU-27
Wheat based Beet based � L-1 � GJ-1 � t-1 � L-1 � GJ-1 � t-1 Net feedstock cost - Feedstock 0.40 18.9 790 0.26 12.3 513 - Co-product credit 0.15 7.1 296 0.03 1.4 59 Subtotal feedstock cost 0.25 11.8 493 0.23 10.9 454 Conversion costs 0.28 13.3 553 0.22 10.4 434 Blending costs 0.05 2.4 99 0.05 2.4 99 Distribution costs 0.01 0.5 20 0.1 4.7 197 Total costs at petrol station 0.59 27.9 1165 0.6 28.4 1184
Source: Biomass Technology Group, 2004 (published on www.eubia.org)
56
Table 7 Engineering cost estimates for bioethanol plants in Germany
Plant capacity 50 million litres 200 million litres Raw Material Wheat Sugar beet Wheat Sugar beet Feedstock cost $0.28 $0.35 $0.28 $0.35 Co-product credit $0.07 $0.07 $0.07 $0.07 Net feedstock cost $0.21 $0.28 $0.21 $0.28 Labour cost $0.04 $0.04 $0.01 $0.01 Other operating and energy costs $0.20 $0.18 $0.20 $0.17 Net investment cost $0.10 $0.10 $0.06 $0.06 Total $0.55 $0.59 $0.48 $0.52 Total per gasoline equivalent litre $0.81 $0.88 $0.71 $0.77
Source: International Energy Agency, 2004; Data Source: F. O. Licht�s, 2003. Price in US $ L-1
3.9.3 Production of chemicals and biopolymers in beet
A report being prepared by Platform Groene Grondstoffen (green raw materials)
(http://www.senternovem.nl/energietransitie/groene_grondstoffen/index.asp) [137]
investigates the prospects of biomass production in the Netherlands for energy and
platform chemicals. It concludes that amongst the various crops considered, sugar
beet is especially promising due to its high biomass yield. While the CAP-reform
has significantly reduced the profitability of sugar beet (in the Netherlands from �
1350 to � 550 per hectare), new co-products such as amino acids or organic acids
overproduced by GM-technology could restore profitability to the farmer to levels
similar as before the CAP-reform. These new co-products (e.g. lysine or itaconic
acid) would allow delivery of beet ethanol for prices between � 0.28 and � 0.35 L-1,
and lysine for prices between � 1000 and � 530 t-1.
3.9.4 Development and registration costs of GM-beet
The production of new feedstocks in beet depends on GM-technology. As
discussed in Chapter 1, this entails significant costs and risks. Many GM-crops fail
57
to reach the market due to technical, financial, regulatory, or societal reasons [72].
The Biotechnology and GMOs website of the EC Joint Research Centre currently
lists 2121 deliberate releases of transgenic plants in the EU, 278 of which involve
transgenic beet (biotech.jrc.it/deliberate/gmo.asp). Most of these field trials are
related to glufosinate, glyphosate and virus resistance. However, a series of
releases are related to the production of amino acids (asparagine, histidine) or
modified sugars (by expressing the enzymes invertase, mannose isomerase, levan
sucrase). The companies involved in these field trials are Van der Have (France),
SES (France), and Novartis Seeds. Presently available are glyphosate and
glufosinate varieties, although the approval of glufosinate-tolerant varieties for
market access has been withdrawn by the applicant [72]. Research focused on
other active ingredients has not led to further herbicide-tolerant varieties. Märländer
has reviewed the economics of genetically modified herbicide-tolerant sugar beet
varieties in Europe and came to the conclusion that total cost savings of � 180
million per year were calculated for the area of 1,700,000 ha in the main EU sugar
beet-growing countries. However, market entry of glyphosate-tolerant sugar beet
was not successful for political reasons and the lack of acceptance of genetically
modified varieties by the consumer [164].
3.10 SWOT analysis
Strengths
! Yield per ha higher than for any other conventional crop grown in Europe
! Adaptation of existing processing facilities is possible
! Established annual crop
! Valuable rotation crop (uses excess nitrogen)
! Great opportunity for breeding, to maximise new uses
! Abundant natural genetic variation, routine transformation, MAS, QTL
! Salt tolerant
58
Weaknesses
! Food and feed crop
! High input crop (energy, fertiliser, pesticides)
! Sugar beet cultivation causes soil compaction, and water / wind erosion
! Outcrossing and transgene spread is a risk
! High harvesting costs due to below ground biomass
! Locked supply chain
Opportunities
! Sugar-reform leads to search for other applications
! Breeding for biomass/fermentable sugar has not been done: great potential
for improvement
! Development of non-food uses for the crop to produce bioproducts
! The demand for biofuel is increasing
! Added value products
Threats
! Sugar-reform may eliminate production and processing capacity before
promising transgenic crops are available
! Sugar beet for chemicals and polymer necessitates transgenic crop
development
! Uncertainty about processing
! Uncertainty about product prices
! Competition with sugar cane
3.11 Research and development needs
3.11.1 General R&D needs
A number of general R&D needs can be identified based on the information
presented in this report. Several general areas can be distinguished
59
Sugar beet cultivation:
! Variants with longer growth season (winter crop), allowing higher yield
and/or extended processing campaigns. This could be accomplished by
improved bolting resistance and winter hardiness [264]
! Reduction of water, energy, fertiliser and pesticide input
! Reduction of soil erosion by reduced tillage cultivation
! For bioethanol production: increased nitrogen improves yield by 5% [264]
Sugar beet processing:
! Prolonged beet storage (longer shelf life), extending the processing period
! Storage of dried beet milled to powder
! Reduced waste and energy input
! Integration of co-product processing
Sugar breeding and genetic or metabolic engineering:
! New breeding targets (away from technical quality, towards higher
fermentable material include sugar and higher biomass)
! More thorough understanding of plant cell metabolism, regulation, transport
and storage
! Prevention of deleterious effects on plant health, yield, and processing
Use of transgenic beet (unavoidable in the production of biopolymers or chemicals):
! Prevention of transgene flow
! Public perception on the acceptance of a GM energy or industry beet
! Safety of transgenic beets with respect to anti-nutrients [10] and other
aspects [170]
3.11.2 Specific ideas from reports and scientific literature
It appears that the production of chemicals and biopolymers in crops such as beet is
still far away due to issues such as cost and timeline of development, limited
knowledge on metabolic engineering in plants, unsolved problems in various areas
60
such as gene flow, yield and processing issues, and not least public acceptance.
Several reports and papers make specific suggestions on potential co-products that
could be obtained from sugar beet [13, 105, 139, 241] (Table 8). Of the ideas that
require genetic engineering, only fructan has been implemented [228].
Biopolymers
Beet contains several biopolymers, such as araban (a polysaccharide composed of
the pentose sugar L-arabinose), pectin (a mixture of linear and branched
polysaccharides containing 1,4-linked α-D-galacturonic acid units, and L-
rhamnopyranose units), cellulose, lignin, and proteins (Figure 3). These are
currently harvested as pulp, and used for feed or fermentation. Beet pulp contains
15-30% pectin with good emulsifying but rather poor gelling properties. Efficient
extraction methods could make this a potentially valuable product [277] if it could be
modified for better properties [139]. Sugar beet pulp contains relatively little lignin,
but good quality cellulose. Purification of this cellulose elimates costly and
environmentally damaging separation steps, especially if overproduction of cellulose
in beet roots can be combined with down-regulation of lignin biosynthesis, as has
been done successfully for several plants [29]. Pure cellulose, which can be used in
hydrocolloids, can be obtained from sugar beet pulp by removing hemicellulose and
pectin using mild alkaline disencrusting and bleaching treatments [74]. Conversion
to carboxymethylcellulose [249], and thermoplastic materials [213, 214] has also
been considered. Efforts to produce new polymers such as polyhydroxyalkanoates
in beet are limited. PHB was produced in beet hairy root cultures [175], thus far
without follow-up. Other biopolymers to consider are polysaccharides derived from
sucrose, non-ribosomal proteins such as cyanophycin, and fibrous proteins such as
silk, elastin, synthetic sequences [221].
61
Chemicals
As sucrose is the main product of sugar beet, it has been suggested to convert this
resource in planta to other useful products. Bacterial enzymes such as glucoside-3-
dehydrogenase could be expressed in the sugar beet and catalyze the conversion
of sucrose to keto-derivates. The product would be useful for detergents or as a
starting point for the synthesis of polyurethanes [139]. Similarly, isomerases may be
used to produce isomaltulose (palatinose) or sorbitol as was successfully done in
sugar cane [92, 274]. Currently, the fermentative production of citric acid from
molasses using Aspergillus or other cultures is cheaper than extracting citric acid
from citrus fruits. These and other plants concentrate the citric acid in the vacuoles,
with the aid of ATP-dependent transport processes. Once the responsible proteins
have been characterized and cloned [233], these transporters could be expressed
in sugar beet, allowing the concentration of citric acid in the sugar beet vacuoles.
The citric acid could then be purified from the press juice [139]. This general idea
can also be extended to other metabolites. Itaconic acid (an replacement for
methacrylate) can be produced from aconitic acid (a naturally occurring compound
in sugar beet) using a bacterial decarboxylase [270]. The feasibility of creating a
glycerol beet has also been investigated (www.mrac.ca/index.cfm/fuseaction/
prj.details/ID/093A3413-E825-1F48-192FAF4F0AA100D8/index.cfm). Sevenier and
co-workers have proposed to increase the nutritional value of crops by specifically
increasing the content of amino acids, vitamins, or other health promoting
substances [229]. One example is a 15-fold increase in lysine content in the potato,
which should also be feasible in sugar beet. All of these ideas require a much
greater understanding of plant cell metabolism, regulation, transport and storage
than is presently available.
62
Table 8 Possible novel products from sugar beet
Product Variety Processing References Chemicals Ethanol (from side-streams)
Current beet varieties Limited changes [75]
Ethanol (from whole beet)
New beet varieties New process [247]
New sugars Transgenic New process Chemicals (general) Transgenic New process [139, 241] Amino acids (co-product of bioethanol)
New beet varieties or transgenics
Melassogenic, thus new process
[229]
Organic acids (citrate, itaconic acid, etc.)
Transgenics Melassogenic, thus new process
[139]
Uridine / other nucleosides
New varieties or transgenic
Molasses processing
[130]
Polymers Fructan or other polysaccharides (dextran)
Transgenic New process, assuming 100% fructan
[228]
Cellulose Transgenic New pulp processing, juice processing unchanged
[139, 213, 214]
Pectin New varieties or transgenic
New pulp processing, juice processing unchanged
[139]
Polyhydroxy-butyrate Transgenic New pulp processing, juice processing unchanged
[175]
63
4 TOBACCO (NICOTIANA TABACUM L.)
4.1 Introduction
Of all crops considered for the production of chemicals, polymers or proteins,
tobacco (Nicotiana tabacum L.) has received the largest share of attention, because
it is easy to manipulate and abundant experience has accumulated. Tobacco is of
special interest as a target for GM, because it is fairly easy to transform the
chloroplast, as opposed to almost any other plant. This has important implications
for the ease with which the metabolism can be manipulated, because expression
levels are much higher and easier to regulate. Moreover, tobacco has significant
biomass potential. As a non-food crop with limited gene flow risks it can also be
considered a safe crop.
Tobacco belongs to the Solanaceae, the same family as the potato, tomato, pepper
and poisonous nightshade. Tobacco was brought to Europe and the North
American colonies by the early explorers of South America and was grown and bred
for smoking and chewing. Believed to be a cure-all, it was used to dress wounds
while chewing tobacco was thought to relieve the pain of a toothache. Cigarettes
became popular in the second half of the 19th century. During and after the 1950s, it
became clear that tobacco smoking is a leading cause of death and illness in large
sections of the population, making smoking and tobacco cultivation a controversial
subject.
In the EU, tobacco is grown in Greece, Italy, Spain, Portugal, France, Austria and
Belgium (Figure 6). Its cultivation in Europe (5% of world production) depends
almost entirely on subsidies, which amounted to � 963,000,000 in the year 2003.
Most of the 350,000 tons of raw tobacco produced in Europe is of dark varieties for
which there is a limited market only [14].
64
Figure 6 Tobacco production in the EU-15. Unit: 1000 tons.
Data for 2001: Belgium, France; data for 2000: Greece, Austria, Portugal; data for 1999: Spain, Italy; data for Germany not available.
The Common Agricultural Policy reform implemented for tobacco in 2004 changed
the basis of existing tobacco subsidies, coupled to production levels, incorporating
them into the single farm payment. Since, in this system, subsidies are decoupled
from production farmers will have more possibilities to farm to the demands of the
market and develop more environmentally friendly systems of production. It was
agreed that the switch from direct support for production to the single farm payment
will be completed and applied in full by 2010. During the interim period at least 40%
of direct aid for tobacco under the old system will be allocated to single farm
payments. From 2010, 50% of the aid for the tobacco sector will be used to
establish a financial allocation for restructuring in tobacco growing areas, as part of
rural development activities. The Community Tobacco Fund also funds education
and information activities to improve public awareness of the harmful effects of
65
tobacco consumption. Future cultivation for non-food applications could, under the
single farm payment scheme, take place on set-aside or non set-aside land.
In contrast to the production of tobacco for smoking purposes there is no
controversy about the role of tobacco as an excellent model plant for science. It was
the first plant to be regenerated to transgenic plants [26] and the first to be tested
extensively for the heterologous expression of foreign proteins [90, 252]. A new
future for tobacco may be found in the large-scale agricultural production of
pharmaceutical proteins, vaccines and industrial enzymes [59, 90, 105, 117, 120,
121, 252]. Based on such considerations, companies such as Planet Biotechnology
Inc. (www.planetbiotechnology.com) and Meristem Therapeutics (www.meristem-
therapeutics.com) have adopted tobacco as a platform crop for the production of
biopharmaceuticals. These and other companies have tobacco plant-derived
pharmaceuticals undergoing phase-II clinical trials [90]
(www.molecularfarming.com).
Whether tobacco is also suited as a crop for the production of biopolymers, platform
chemicals, and other products such as enzymes, depends on many factors. The
main impetus for production in plants is that it is � theoretically � much cheaper than
production in bacteria or yeast. The latter route is relatively straightforward and well
established, but requires significant inputs in the form of energy and raw material.
Presently, there are few concrete examples of tobacco-based biopolymer or
chemical production. None of these projects has reached the commercial or pre-
commercial stage, and in most cases the most convenient varieties of tobacco were
selected for the project, not the variant or hybrid that would be most suited for
production. Considering the different demands on biomass properties, the
production of polymers and chemicals requires the selection of Nicotiana species or
hybrids that have quite different growth properties than N. tabacum. In addition,
these bioproduction variants will be grown under conditions that may differ
considerably from standard tobacco cultivation.
66
The large-scale processing of plant material to release a polymer or chemical as an
issue that may make or break the project is discussed only in few scientific papers.
Finally, the regulatory framework is quite important as the EU now subsidizes
tobacco growers to produce a crop of a quality that has a limited market in the EU
and beyond. New uses for tobacco may ultimately provide a way out of the current
situation both to growers and the EU. As an easy to manipulate non-food crop
without close wild relatives in Europe and North America it also has great
advantages regarding safety and possibly also public acceptance of GM-crops.
4.2 Current and future co-products
4.2.1 Biopharmaceutical proteins and vaccines
As tobacco is a well-established expression host for which many tools and
procedures are available, it has been the favourite host for molecular farming from
the early days of plant biotechnology [59, 160]. Drivers range from easy scalability,
cheap production, absence of human pathogens, easy storage (of seeds) and the
ability to carry out post-translational modifications. Several biopharmaceuticals have
successfully been produced in tobacco, including antibodies (to 0.5 g per kg leaf)
[101], and cytokines such as interleukin-10 [172] (for a review of plant-produced
biopharmaceuticals see [159]; for plant-produced vaccines see [210]). The website
www.molecularfarming.com lists 9 tobacco produced vaccines and
biopharmaceuticals undergoing clinical trials. In Europe, most field trials were
carried out using tobacco (all by Biochem/Limagrain), whereas in other regions and
especially in the USA, tobacco plays a relatively minor role (www.gmwatch.org). A
large part of the research in Europe in this area is coordinated by the FP6 program
Pharma-Planta (www.pharma-planta.org).
One factor limiting the use of tobacco is the presence of toxic alkaloids, preventing
the direct use of plant material as an edible vaccine or treatment (although low-
alkaloid tobacco is available [172]). Thus, many groups have started to consider
other crops as hosts, such as lettuce, carrot, spinach, alfalfa, potato, maize, etc.,
67
especially when direct oral administration of the biopharmaceutical is desirable.
However, all of these crops are food or feed crops, and inadvertent entry in the food
chain is a risk with potentially harmful effects on human health [159]. This is not an
issue for tobacco.
4.2.2 Industrial enzymes
Enzymes are increasingly used in industrial processes, and the price of enzymes is
an important issue. For example, current political developments (10% biofuels in the
EU in 2020) are likely to create a very significant market for cellulases (several
billion Euros if the enzymatic route prevails), but only if the price of the enzyme can
be lowered significantly from 30 cents per gallon to 5 cents per gallon of biofuel
[207, 243]. One approach is to express the cellulase in a biomass plant such as
maize, and activate the enzyme in situ after harvesting [35]. Cellulase has also
been expressed to levels of up to 1% in the apoplast of tobacco [61, 281]. Simple
processing by homogenizing the leaves could be sufficient to provide a cheap
enzyme preparation. Industrial interest is evident in news releases from companies
such as Syngenta, which collaborates with Diversa on the production of enzymes
including cellulases in plants (www.allaboutfeed.net), and the recent merger of
Diversa with Celunol, one of the cellulosic ethanol frontrunners (www.celunol.com).
No fundamental barriers stand in the way of efficient and high-level protein
production in plants, and levels of up to 47% of total leaf protein have been reported
[64, 67]. The xylanase xynA gene could be expressed from the tobacco chloroplast
genome to levels of 6% total soluble protein, corresponding to an activity of about
140,000 U kg-1 of fresh leaf tissue. Because the enzyme is thermostable, it can be
purified by the use of heat in the first step of purification. This also reduces
proteolytic degradation by denaturing host proteases. Moreover, more than 85
percent of the xylanase activity seen in fresh leaves was still present in sun-dried or
even in senescent leaves. The enzyme did not cause cell wall degradation when
expressed in chloroplasts, as opposed to nuclear transformants [141].
68
A bacterial 4-hydroxyphenylpyruvate dioxygenase, which provides strong herbicide
tolerance to tobacco, could be expressed to at least 5% of the soluble mature leaf
proteins in transformed tobacco [78], and a study directed at the production of p-
hydroxybenzoate (see below) reported the overproduction of the E. coli enzyme
chorismate pyruvate lyase from the tobacco chloroplast genome to 30% of total
soluble protein [260]. Further examples of technical enzyme production in tobacco
have been reviewed elsewhere [34].
4.2.3 Polymers
Efforts to produce polymers in tobacco have concentrated on protein polymers such
as fibrous proteins (silk, elastin, and synthetic sequences) [173, 223], non-ribosomal
proteins such as cyanophycin [57, 221, 222], and polyhydroxyalkanoates (PHAs)
[239]. These studies show that production of such materials in tobacco is feasible
but not trivial. Up to 2% spider-silk protein of total soluble protein (TSP) could be
accumulated in the endoplasmic reticulum (ER) of tobacco leaves using various
synthetic spider-silk genes (see references cited in [221]. Purification of the silk
protein is greatly facilitated by the heat-stability and resistance against acidification
of silk proteins [223].
Expression of the cyanophycin synthetase-coding region from the cyanobacterium
Thermosynechococcus elongatus yielded both water-soluble and water-insoluble
forms of cyanophycin to a maximum amount of 1.14% of dry weight in tobacco
leaves. Growth of the transgenic tobacco was affected, which was attributed to the
depletion of amino acid resources [188]. Expression in the chloroplasts of
transgenic potato is claimed to reduce stress and significantly increase yield (Inge
Broer, personal communication).
Production of PHAs by means of genetic engineering of green plants has long been
thought to reduce the production costs to economical levels [202]. This has led the
US company Metabolix to initiate a project to produce polyhydroxybutyrate in
switchgrass. In tobacco, almost no polyhydroxybutyrate (PHB) could be detected
69
when production in the cytosol was attempted [184, 186], in contrast to similar
studies in Arabidopsis [187]. Plastid-based accumulation of PHB in tobacco was
also much lower than in Arabidopsis. Direct transformation of the tobacco plastid
genome yielded levels of up to 1.7%, but was accompanied by male sterility and
growth deficiencies [154, 155]. Production of medium chain length PHAs (mclPHAs)
in tobacco plastids also yielded only low levels (0.5% of leaf dry weight) [262].
These studies suggest that a much better understanding of (sub)cellular metabolism
and metabolic engineering, and the causes of deleterious effects of polymer
production on plant growth and health, is required to enable more promising results.
Although industrial interest is evident by a multitude of patents and publications,
commercial applications appear distant.
4.2.4 Platform chemicals
A clear demonstration of the feasibility of feedstock production in tobacco is the
production of p-hydroxybenzoic acid (pHBA), the main monomer used in liquid
crystal polymers. It can be synthesized from chorismate by the E. coli enzyme
chorismate pyruvate lyase (CPL), which is encoded by ubiC. In plants, the
conversion of chorismate to pHBA also occurs naturally, but at low levels and it may
require up to ten successive enzymatic steps. When ubiC was expressed in nuclear
transgenic tobacco, the glucose conjugates of pHBA (phenolic glucoside and the
ester) accumulated to 0.52% dry weight (dw) [235]. Expression in chloroplast
transformed tobacco yielded much more of the pHBA-glucose conjugates, reaching
a maximum of 15% dw after 100 days in soil. Under continuous light levels of 26.5%
were reached, which was claimed to be high enough for commercially viable
production. Both the phenolic glucoside and Glc ester are produced in the cytosol
and are subsequently transported into the vacuole by different carriers [260]. CPL-
expressing plants did not show deleterious effects and were perfectly healthy
despite the massive diversion of chorismate to pHBA. This clearly indicates that the
flux through the shikimate pathway in plants is quite flexible and is still capable of
providing for essential downstream intermediates such as phenylpropanoids. A
70
challenge for a commercially viable, plant-based pHBA production platform is to
control the partitioning of pHBA Glc conjugates. While the phenolic conjugate is
quite stable, the ester, however, is acid and base labile. Since the compound of
interest is free pHBA, partitioning of most pHBA to the ester is preferred [260].
As the aromatic amino acid synthesis pathways seem amenable to manipulation,
the anthranilate synthase gene ASA2 encoding for an α-subunit insensitive to
feedback inhibition by tryptophan was cloned and expressed in transgenic tobacco
chloroplasts [280]. Transgenic plants exhibited increased expression of the AS α-
subunit and a 4-fold increase in AS enzyme activity, resulting in a 10-fold increase
in free tryptophan in the leaves. The approach has also been used with several
plants such as rice, azuki bean and potato [106], generally aiming to improve the
nutritional value of the crop.
Tobacco plants were also modified to produce (+)-limonene [157]. Subsequent
hydroxylation of (+)-limonene to (+)-trans-isopiperitenol was accomplished by co-
expressing a P450 hydroxylase [156]. Chappell and co-workers engineered tobacco
to increase the level of patchoulol and amorpha-4,11-diene (the immediate
precursor to the anti-malaria drug artemisinin, which is in short supply) more than a
1000-fold [275]. As these compounds are volatile, and are lost during growth of the
plant the authors propose that the introduction of glycosyltransferases would
significantly increase yields [41]. Tocotrienol (vitamin E) production up to 0.4 mg g-1
dry wt (most of it the preferred isomer α-tocotrienol) was obtained by the co-
expression of the yeast prephenate dehydrogenase gene and the Arabidopsis p-
hydroxyphenylpyruvate dioxygenase coding sequence [212]. Total sterol content
could be increased 6-fold to almost 10 mg g-1 dry weight by simple expression of
the Hevea brasiliensis hydroxy-3-methylglutaryl-Coenzyme A reductase-1 [220].
Several sugar derivates that normally occur at low concentrations in plants may be
overproduced through the heterologous expression of enzymes such as
isomerases, transferases, and dehydrogenases. A sucrose isomerase from Erwinia
rhapontici expressed in transgenic plants produced up to 45 times more
71
isomaltulose (IM) than sucrose, but showed severe phenotypic alterations and
strongly impaired growth. This was probably due to the fact that IM accumulated in
all cell compartments instead of only in the vacuole [37]. A comparable study where
sugar cane was modified to produce IM and accumulated in the vacuoles was very
successful: as much IM as sucrose was produced at unchanged levels of sucrose,
thus essentially doubling the sugar content [274].
Transgenic tobacco plants that accumulate polyols (levels up to 0.5% of wet weight)
often show growth inhibition, necrotic lesions or other deleterious effects (see
references cited in [69]). However, trehalose, used in the pharmaceutical industry
as a preservative and highly toxic when produced in the cytoplasm, was non-toxic
when accumulated within plastids: chloroplast transgenics accumulated 15�25 fold
more trehalose than the best nuclear transgenic plants [140].
Thus, chemicals can be overproduced in tobacco to very significant levels already
with a limited number of changes (added genes, localization, regulation). Efforts
comparable to the metabolic engineering of microbes are likely to yield plants
suitable for commercial production.
4.2.5 Plant oils
It has been suggested that tobacco seeds could be exploited as a potential source
of a useful plant oil. Several properties of this oil, such as viscosity, energy content,
flash point and density are very similar to those of other plant oils [100]. Other
properties, however, such as cloud point and pour point, differ significantly. Tobacco
seeds contain up to 40% of oil that is very high (75-78%) in linoleic fatty acid (18:2),
and high in oleic (11%) and palmitic (9%) fatty acids (18:1 and 16:0). This is
reflected in the high pour point, which for tobacco oil is -14° C, while rapeseed and
corn oil have pour points as low as -32° C and -40° C respectively. Tobacco oil could
be a suitable feedstock for the production of biodiesel [100]. However, it is
questionable whether this crop should be promoted as a potential non-crop platform
72
for production of industrial oils. Although each plant produces roughly 30 g of seeds,
the seeds are extremely small (1 g equals 10,500 seeds), which complicates
handling. Moreover, oil production per hectare is low. In traditional cultivation
(20,000 plants ha-1, [238]) and assuming an oil content of up to 40%, tobacco would
only produce 240 kg ha-1. At higher densities, such as used in biomass production,
more oil could be produced. However, here the biomass is harvested before the
tobacco flowers and sets seed. For comparison, commercial cultivars of oat, which
is not yet considered as an oil crop, produce an oil yield of 550 kg ha-1, and
established oil crops such as rapeseed and sunflower produce up to 1.1 t ha-1.
4.2.6 Processing of tobacco for biopolymer and platform chemical production
Few publications dealing with the production of biopolymers or specific compounds
in tobacco focus on the processing method that is required to release and purify the
target compounds, although the various options are discussed to some extent. The
different steps shown in Figure 7. (adapted from [121]) can be taken as a general
flow-scheme of a processing strategy. In the EPOBIO project, tobacco was chosen
as a representative of the leafy plants: the biopolymer or platform chemical to be
produced is most likely located in the cytoplasm, in the chloroplast or in the
vacuoles, either in a dissolved state (chemicals, soluble proteins) or in the form of
discrete granules (most polymers, insoluble proteins). This has consequences for
harvest, storage and processing, because some compounds may readily degrade
during these steps. In the future, biopolymers and platform chemicals produced in
GM-tobacco will be purified in a biorefinery setting.
Typically, wet separation techniques will be used for crops that contain high
amounts of internal water and water-soluble protein. Protein concentrates and
isolates can be obtained without an active solubilization process: simple disruption
of the cells and squeezing yields a protein-rich juice. For specific enzymes, such as
cellulases used in non-food processes, it may be sufficient to simply grind up the
leaves, removes solids, and stabilize the leaf juice by changing the pH and salt
concentration, and addition of inhibitors, reducing agents, etc. [73]. In some cases,
73
special care will have to given to the removal of polyphenols, alkaloids (especially
nicotine is a highly toxic compound), and other problematic substances. However, in
part this problem can be solved by breeding, or by choosing an appropriate
Nicotiana hybrid or species [172]. In addition, young tobacco plants contain much
less alkaloids.
Cult ivat ion
Harvest ing
Transport at ion
St orage
Tissue processing
Ext ract ion
Purif icat ion
Target compound yieldConf inement requirement sGeogrpahical limitat ionsSeasonal limit at ions
Mechanical propert iesTime sensit iv it yMoisture sensit iv it y
Temperature sensit iv ityMoisture contentDensity
Temperature sensit iv ityCompound st abilit y in t issue
Stability in t issueEnrichment methodTissue mechanical propert iesCo-product s
StabilityConcent rat ion
Interfering agent sRecycling
Figure 7 Processing steps and issues in the production of chemicals from plant materials
74
For polymers that accumulate as granules, it may be possible to use methods
similar to starch processing from potato or maize. Hydrophobic polymers may be
extracted using solvents, although this is likely to be an expensive option. For
proteins, one may consider current methods to recover protein from waste streams
such as is common practice in the potato processing industry, or in the isolation of
protein from grass [73].
In the case of biopharmaceuticals, more thought has been given to the issue of
protein recovery from leaf material [174]. The recovery of biopharmaceuticals from
tobacco leaves is generally started by blending and homogenizing the fresh leaves
for protein extraction, for example by aqueous two-phase extraction [24]. However,
it remains to be seen if methods developed to recover high-price
biopharmaceuticals can be adapted to bulk-scale proteins. The recovery of
chemicals will very much depend on their chemical and physical properties.
Details on processing setups tested to isolate ribulose 1,5-diphosphate carboxylase
and other proteins from tobacco can be found in a range of publications from the
1980s [230-232, 267, 268, 273].
The waste streams from processing tobacco are likely to contain relatively little dry
mass, and would probably be suited for the production of biogas using existing
technology. The production of ethanol has also been proposed [76]. The
composition of tobacco biomass is:
! Sugars: 20-28% - sucrose, levulose, and other free reducing sugars that are
easy to ferment
! Starches: 8-14%
! Cellulose: 30-45% - of which 85-90% is holocellulose with minimal lignin
encasement, thus easy to ferment
! Lignin: 1.5 � 2.0%
! Proteins: 20% - most of which can be broken down in Fraction 1 and
Fraction 2 protein
75
Most of the material is claimed to be easily fermentable, although no report has
appeared in scientific sources.
4.3 Genetics
N. tabacum L. is an allotetraploid cross between N. sylvestris (2n=24) and N.
tomentosiformis (2n=24), both species native to the Argentina-Bolivia border region.
The tobacco nuclear genome is very large, in the range of 4500 megabasepairs (1.5
times the size of the human genome) and is being sequenced under the Tobacco
Genome Initiative project at the College of Agriculture and Life Sciences, North
Carolina in collaboration with Orion Genomics, St. Louis, Mo. In January 2007,
80,000 ESTs and 1,700,000 individual clones were sequenced (www.intl-
pag.org/15/abstracts/PAG15_P05g_449.html). Orion's GeneThresher technology is
used to develop an overall map of the tobacco genome that will identify up to 90
percent of tobacco's genes. A BAC library with 9.7-fold genome coverage of N.
tabacum genome has been constructed. BAC end sequencing and construction of a
physical map is in progress (www.ncbi.nlm.nih.gov). The tobacco chloroplast
genome was sequenced already in 1986 [234].
A summary of tools and methods in tobacco biotechnology is given in chapter 3 of
Tobacco: Production, Chemistry and Technology [42]. Most of these methods can
be considered part of general plant biotechnology, and need not be discussed in
detail. Many of the new developments after 1999 relate to foreign gene expression
in the chloroplast, as discussed below. An outstanding feature of tobacco is that
chloroplast transformation was first developed in this crop, and is routine only in
tobacco, and to a lesser extent in other solanaceous plants such as potato and
tomato [162]. Many of the studies cited in section 4.2 indicate that chloroplasts
provide an ideal site to accumulate proteins or biosynthetic products that are
harmful if they are localized in the cytoplasm [36, 64].
Chloroplast transformation is an excellent method for expressing foreign genes in
plant cells. As all genomes of the up to 100 chloroplasts within the cell are
76
transformed, and each chloroplast contains about 100 copies of the chloroplast
genome, the gene copy-number is very high compared to transgenes in the nuclear
DNA. This increases the amount of protein produced in a single cell significantly.
Typically, chloroplast transgenic lines express similar levels of foreign proteins,
within the range of physiological variations, and gene silencing by co-suppression
has not been observed to occur [65]. Chloroplasts are derived from a free-living
oxygen-producing photosynthetic prokaryote. Thus, plastids handle genetic
information in ways very similar to modern prokaryotes. The plastid genome
contains operons typical for such organisms; thus heterologous genes can be
introduced in the plastid genome as polycistrons [15], and only one promoter and
one terminator is required to control all new genes. Transit peptide sequences are
no longer required, and regulated expression is greatly facilitated, making elaborate
metabolic engineering a realistic option. New tools are being developed for
regulated expression in plastids [154, 181]. For example, a hybrid transcription
system was devised that introduces a new RNA polymerase for selective
transcription of transgenes [46].
What has to be recognized is that - as has been the case for bacteria - many
different constructs, promoters, terminators, and signal peptides may have to be
tested to obtain significant, reliable, well-regulated expression under the required
conditions in the field. Far fewer studies deal with the expression of proteins in
plants than in simple bacteria, and many efforts were held back or cancelled due to
concerns on biosafety. An effort of the same magnitude as with bacterial expression
systems will undoubtedly bring many goals in sight.
4.4 Breeding
Tobacco (N. tabacum L.) belongs to the family Solanaceae, which contains several
well known cultivated crops such as tomato (Lycopersicon esculentum), eggplant
(Solanum melogena), pepper (Capsicum annuum) and potato (Solanum
tuberosum). The natural distribution of the genus Nicotiana is limited to America (75
%), Australia and a few islands of the South Pacific (25 %). The estimated 60
77
species of Nicotiana are classified into 14 sections based upon distribution and
morphological and cytogenetic characteristics [102].
For an overview of current tobacco breeding methods we refer to [142]. Germplasm
collections are located in the USA and several other countries. These tobacco
introductions and the wild Nicotiana species are valuable sources of genes for
disease resistance and other traits. Induced mutations are another option, except
that most such changes are deleterious and recessive. Crosses among lines of the
same class of tobacco are easiest, while utilization of germplasm from wild
Nicotiana species is complicated and time-consuming.
For several centuries tobacco has been selected for desirable traits such as
improved quality, ease of handling, filling ratios, aroma, and flavours. Several types
of traits have been investigated in great deal, and many of these traits (yield, plant
morphology, disease resistance, insect resistance yield) [142] are also relevant for
feedstock production, where biomass and product yield are the primary goals.
As it cannot be predicted which Nicotiana selection, species or hybrid will be most
suitable for a specific application, we will not discuss these traits in detail. However,
some of the common cultivars (e.g. dark fire-cured varieties) produce much higher
biomass yields than others (e.g. variety Maryland) [230], providing a solid base for
further selections. New methods, such as metabolic profiling for biomass yield and
growth rate [176], are certain to make a significant contribution as well.
A number of morphological traits are of outstanding interest if tobacco is to be
considered as a potential biomass producer in parallel to its use for high-value
products. The indeterminate growth habit (Mammoth) implies that plants continue to
grow until late summer; the short-internode trait means that more leaves per unit
stem length are produced; while the wrinkled or ruffled leaf surface traits may
influence biomass yield or processing [142]. Some traits are specific for smoking
purposes, and would have to be eliminated or simply become irrelevant. For
example, the low chlorophyll trait in white burley types probably negatively impacts
78
biomass yield. The amount of alkaloid and leaf surface compounds is also
important, as waste-biomass might find use as feed components: the amount of
these compounds should be reduced. In the case of alkaloids, two qualitative
genetic loci are involved. Plants with two recessive alleles produce only 0.3 %
alkaloids, while wild-type lines contain 4.6 % [142].
The Kentucky Tobacco Research and Development Center (KTRDC) is developing
new Nicotiana hybrids for higher biomass yield, especially in the context of using
tobacco as a production system for biopharmaceuticals. In these studies, the
maternal line was usually a cultivar of N. tabacum modified and/or bred to produce
a product of interest, while the paternal line was another Nicotiana species such as
N. Benthamiana, N. glauca, N. glutinosa, N. quadrivalvis, N. otophora or N.
sylvestris. The interspecific hybrids typically were vigorous plants that are more than
99% male and female sterile [279]. The latter is of advantage to prevent
transmission of foreign genes to related crop species. However, it also means that
the crop cannot be established by sowing. Many Nicotiana interspecific hybrids
express lethal symptoms, especially in case of hybrids involving N. tabacum [246].
Most other hybrids, such as those with N. glauca are sterile [250].
As tobacco is an established crop, many conventional breeding tools and
experience are available. Nevertheless, the number of publications on molecular
tools such as quantitative-trait-loci and marker-assisted selection is relatively
limited. Most of these focus on resistance to pathogens (RAPD and SCAR for
resistance to blue mould; RAPD for resistance to black shank and black root rot;
transfer of a gene conferring resistance to Potato Virus Y using MAS) [23, 129, 150,
177, 189, 258].
4.5 Susceptibility to abiotic stresses
This topic is not specific to biopolymer or platform chemical production in tobacco.
For a general discussion we refer to [66].
79
4.6 Susceptibility to biotic stresses
This topic is not specific to biopolymer or platform chemical production in tobacco.
For a general discussion we refer to [66].
4.7 Agronomy
Several chapters of the standard volume �Tobacco: Production, Chemistry and
Technology� [66] are devoted to various aspects of tobacco agronomy and
physiology. Seed production will probably be very similar for tobacco used in
smoking and for biomass and co-products [126]. However, field practices will be
completely different, depending on the purpose of growing the crop (for smoking
purposes this was reviewed in [91]). While for biopharmaceuticals production in
greenhouses may be most appropriate, biomass production demands cheap and
efficient growth and harvesting regimes [268]. The following section briefly
describes the conventional procedures to produce tobacco, omitting details of the
cultivation and cultivation conditions that are specific for tobacco used in smoking.
At the end of the section, the limited amount of information that is available on the
cultivation of tobacco for biomass and bioproducts is discussed.
Tobacco cultivation for smoking purposes
Tobacco for smoking purposes is started in a closely spaced seedbed, and the
seedlings [238] are transplanted into the field at a density of about 20,000 plants per
hectare. Normally, these plants are grown to a height of 60 to 75 cm, and average
annual yield is around 12.5 t ha-1 fresh biomass. When tobacco is planted for
conventional purposes, it is planted in carefully spaced rows, and each plant is
virtually hand-tended. Under these conditions there is no display of coppicing
behavior, because the plants are never cut back. Instead, tobacco plants have their
secondary growth removed, and their flowering tops when they begin to spike.
80
Tobacco is grown under a wide range of climates but requires a frost-free period of
90 to 120 days from transplanting to last harvest of leaves. Optimum mean daily
temperature for growth is between 20 and 30°C. The crop is sensitive to water-
logging and demands well-aerated and drained soils. Commonly used soils for
tobacco production are sands, loamy sands, sandy loams, sandy clay loams, fine
sandy loams and clay and silt loams. The optimum pH ranges from 5 to 6.5. Leave
quality is affected by soil salinity. Depending on the type of tobacco, fertilizer
requirements vary and in general are 40 to 80 kg ha-1 N, 30 to 90 kg ha-1 P and 50
to 110 kg ha-1 K.
Crop rotation after one or two seasons is recommended, using crops such as grass,
sorghum, millet and maize that are not susceptible to root eelworm. In the US, some
farmers use a one year rotation scheme where small grains are used as cover crop
after tobacco cultivation, followed by forage or row crops in the subsequent year.
The cover crop also tends to prevent soil erosion and returns nutrients and organic
matter to the soil. Most farmers, however, use a three or more year rotation. Most of
the above will probably be valid also when tobacco is grown for biomass and co-
products.
Tobacco grown as a biomass crop
For industrial purposes and as a biomass crop tobacco should be managed more
like a forage crop [76]. Similar to hardwood trees, tobacco will coppice or resprout
from its stump after it has been cut. Coppicing makes multiple harvests in a year
possible, leading to potentially very high yields under the right conditions.
Direct sowing by broadcasting on a conventional plant bed culture system would
enable the farmer to establish stands of between 370,000 and 3,700,000 plants
ha-1. These plants are allowed to grow to a height of about 45 to 50 cm. The crop is
then harvested by mowing with a conventional sickle-bar mower, leaving 15 cm
stumps appearing like a level green table. Within days this flat green surface
becomes a dense tangle of new shoots and leaves of the young tobacco plants.
81
These fresh shoots are at the most sugar-rich and highest protein stage of the
plant's growth cycle. Freshly harvested tobacco has a moisture content of 80-90
percent, leaving 10-20 percent dry weight. This has not been investigated
systematically
Wildman, 1979, estimated that a growth season of 6-8 months would lead to yields
of 165 tons of fresh tobacco leaves [268]. In 1981, Woodlief and co-workers
reported a biomass yield of 74 t ha-1 from three successive harvests, however,
under nitrogen limited conditions [273]. In a similar study, a single harvest in the
middle of June yielded 56.7 t ha-1 [230]. Finally, R. C. Long reported in 1984 that 3
to 5 cuttings per year of a crop started at the highest plant density yielded 140 t ha-1
wet biomass containing 10% dry matter. This biomass contained up to 3 t protein of
which approximately half was extractable, food grade protein. In these experiments
the high plant population led to loss of many plants due to injury and disease.
Equivalent biomass and protein yield could be attained by starting the season with
about 250,000 plants ha-1, in rows spaced 10-30 cm apart [152]. Again, this very
limited number of experiments indicates that breeding and developments in
agronomy will allow a significant increase in yield. However, costs cannot be easily
estimated.
4.8 Environmental impacts
4.8.1 Agrochemical inputs, nutrient and water requirement
Current agronomic procedures for tobacco production are based on the demands of
the tobacco industry. This has led to the following data on tobacco cultivation as
summarized in Table 9.
Nutrients, pesticides and weed control
Tobacco requires large quantities of NO3- fertilizer to guarantee good yields.
Nitrogen fertilization increased the shoot/root ratio [215], and induces higher CO2
82
assimilation rates and leaf area index (LAI) enabling higher yields [58]. However,
over-fertilization of tobacco may result in high concentrations of NO3- in the leaves,
which affects quality for tobacco production. Differences in nitrogen uptake and
subsequent utilization of this nutrient for foliar biomass were found among tobacco
cultivars [216].
Table 9 Cultivation of tobacco related to its environmental impact.
Crop parameters References Plant dry weight 79 g [215] Root density 3.0 cm cm-3 [215] Leaf area/root density
49 dm2 cm-1 cm-3 [215]
Yield 3 t ha-1 to 4.8 t ha-1 [58, 215] Water Water needs 3500 to 4000 m3 ha-1 [215] WUE
0.73, 0.89 1.03 kg m-3 for irrigation at respectively 120, 80 and 40% ET
[215]
Fertilizers Fertilizer need Depending on soil type. Maximum 160 kg ha-1
nitrogen; 60 kg/ha (Umbria, Italy). 40 to 80 kg ha-1 N, 30 to 90 kg ha-1 P and 50 to 110 kg ha-1 K (FAO Cropwatch)
[215]
NUE (mg N) 20.8 to 48.1 [216]
LAI: Leaf area index; WUE: water use efficiency; NUE: nitrogen utilization efficiency
= dry matter of harvestable product per total N; ET: evapotranspiration
Tobacco is a pesticide-intensive crop, ranking sixth in terms of the amount of
pesticides applied per ha [U.S. Government Accounting Office (GAO) 2003] [168].
Common tobacco diseases include root-knot nematodes, black Shank, Blue mould,
Brown spot, Fusarium wilt, Soreshin, target spot, angular leaf spot, Granville wilt,
hollow stalk, TMV, Tobacco Etch, PVY and Tomato Spotted Wilt Virus (cite book).
Several of these diseases can be treated with chemicals. As indicated by McDaniel
et al. (2005) the tobacco industry regards pesticides as essential to an economically
sound tobacco production [168]. Black root rot (Thielaviopsis basicola) is generally
83
severe in soil pH >5.6, but is suppressed under more acidic conditions (pH<5.2).
Soil fertilizers containing NH4+-N (rather then NO3
--N) that have an acidifying effect
on the soil, are generally recommended for burley tobacco production (in North
Carolina). Mineau and Whiteside (2006) showed that the risks of insecticides used
in tobacco cultivation to birds is considerable [178].
Weed-control is essential for conventional tobacco cultivation, but cultivation of the
soil leads to soil erosion. No-till systems would allow production on sloping fields
whilst decreasing the soil erosion. However, weed control in these no-till systems
would be largely chemical and herbicide options are limited in tobacco [83].
Water
Tobacco is very sensitive to excessive soil moisture and is generally considered a
drought tolerant plant. Most of the time irrigation is only needed in dry periods and
only during specific periods in crop development. The water requirements (ETm) for
maximum yield vary with climate and length of growing period from 400 to 600 mm
(from http://www.fao.org/).
For tobacco the calculated seasonal evapotranspiration (ET) is 38 cm, which is low
compared to the 124 cm of sugarcane [266]. At average environmental and crop
conditions, the net irrigation requirements to satisfy the ET in 80% of the crop years
(NIR) is 17 cm [266]. Increasing soil salinity forms a problem in many agricultural
areas. Tobacco is a crop of intermediate tolerance to salinity and tolerates saline
conditions better than oilseed crops, grain legumes, cereals and cotton. While
transpiration rates were unaffected by salinity, plant dry matter and plant height at
harvest decreased, and dry matter partitioning into the leaves increased relative to
the stems [236]. High chloride concentrations in soil water causes nutritional
imbalance and thereby affect the yield and quality of tobacco leaves [182].
Differences in salt tolerance exist among cultivars and choice of the most
appropriate genotype is important for production in saline environments [8]. It is
84
unknown how irrigation and salt stress affects product yield when tobacco is used
for the production of biomass or industrial feedstocks.
Soil
Conventional tobacco cultivation needs intensive tillage, which increases the risks
of soil erosion and decreased soil fertility due to depletion of nutrients and run-off. In
tobacco cultivation, the soil pH should be maintained at pH 5.5 to 6.0 to encourage
root growth and prevent toxic levels of aluminium and iron and to increase the early-
season availability of phosphorus [66]. Furthermore, sub-soiling may improve root
growth in compact soils, whilst a well-developed root system will decrease soil
erosion. No-till transplanted tobacco may significantly reduce negative impacts on
soils [278].
Agrochemical inputs, nutrient and water requirements for tobacco grown as a biomass and bioproduction crop
Cultivation for biomass and bioproducts will significantly change crop demands. The
estimated yield of tobacco or new Nicotiana hybrids to produce chemicals, proteins
or polymers is at least 10 times higher than the yield of tobacco for smoking
purposes. For example, for tobacco biomass production the application of at least
110 kg N ha-1 was recommended to maintain high protein yields [152]. Also,
plantations for biomass will be established by direct sowing (by broadcasting or row
seeding) on a conventional plant bed culture system, not by transplanting seedlings.
It is obvious that this will affect the cultivation conditions (optimal soil and climatic
conditions), all inputs (nutrients, pesticides, herbicides, insecticides, water and
labour), as well as the environmental impact (water requirement, effects on soil,
CO2 emissions and carbon sequestration).