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QLK5-2000-30962
SPINDIGO: Sustainable production of plant-derived indigo |
| Proposal No: | QLK5-2000-30962 | |
| Source: | First Annual Progress Report September 2001 | |
| Second Annual Progress Report December 2002 | ||
| Third Annual Progress Report December 2003 |
Third Progress Report Summary
Period Ending December 2003
Summary
Activities
The project aims to introduce indigo-producing crops into the agricultural systems of the EU so that naturally sourced indigo will supply at least 5% of the European indigo market by 2005, and continue to increase market share, to the long-term benefit of European farmers. In the first three years of the Project indigo-yielding crops have been grown in Spain, Italy, Germany, UK and Finland. The crops have been woad (Isatis tinctoria), Chinese woad (Isatis indigotica),), and polygonum (Polygonum tinctorium),).
Using the key yield parameters of leaf weight and indigo yield per unit leaf weight, genetic lines from a variety of sources have been compared and the relationship between indigo yield and the main environmental parameters of air temperature, global radiation, day-length, rainfall, and photosynthetically active radiation (PAR) analysed.
It was found that woad grows well in all the climatic zones examined, with yields of indigo up to 100 kg/ha. Chinese woad gives higher yields, but is less reliable because of bolting and disease susceptibility; while polygonum yields well,and is more suited to Central Europe and the Mediterranean, but tolerates neither drought nor very high temperatures.
To maximise economic yield for both these crops, the optimum times for sowing, and the optimum times for harvest (up to four harvests per year are possible) have been identified for each climatic zone. Other agronomic factors investigated over the three years are nitrogen fertiliser rates, irrigation requirements, row spacing, and herbicide treatments.
Using traditional breeding methods high-yielding strains of woad have been selected.
An HPLC-based technique for measuring the indigo precursors in woad and polygonum has been developed, and is (at the time of this report) in the process of publication as follows:
The factors that determine seed production such as vernalisation and day-length, have been identified, and sufficient seed can now be produced within the Project for foreseeable future crops of woad and polygonum.
Mobile farm-based extraction machinery designed to handle a high throughput of woad has been developed. Woad and polygonum have also been extracted using non-specialised equipment. Both yield and purity require improvements for the maximum economic benefits to be gained. Novel technology for extraction is also being developed.
Indigo produced in the Project has been used in test dyeings to DIN standards with natural fabrics and unconventional materials. The results compared well with the synthetic product and the present limited commercial sources of natural indigo. In line with the value of SPINIGO indigo as a natural, sustainable product, natural auxiliary agents for dyeing have been identified. However, natural does not necessarily mean sustainable; and to back up sustainability claim, an Environmental Impact Assessment (EIA) of the whole process of natural indigo production has been carried out in draft form.
Benefits and beneficiaries
The progress made in the first three years of the Spindigo Project means that collectively the Project Partners can now claim to have a greater concentration of expertise on natural indigo production than can be found anywhere else in the world.
In Year 1 of the Project SPINDOGO natural indigo was used by the London-based fashion house, Boudicca, for designs launched at London Fashion Week. This early success was followed up in Year 2, when Karada, a leading Italian fashion house used this indigo in a new line (Corpo Nove) of natural fibre men's jackets. lnYear 3, the Italian knitwear designer, Mely's Maglieria, chose this indigo for their new Kos Collection (.koscollection.com), and this indigo was chosen to dye materials for the International Exhibition Grune Woche in Berlin in early 2004.
The messages receive from the fashion and textile industries is that:
'if the means to produce sufficient natural indigo of a high enough quality from European growers can be developed, then the market is there'.
Future Actions
In the final period of the project (2004) the knowledge gained by producing Agronomic Blueprints for the indigo crops will be passed on. In both a web and printed format, these will advise farmers and growers on crop cultivation and indigo extraction. They will also help pass on the message of environmental responsibility and sustainability embodied in the Environmental Impact Assessment that will be completed.
The final year will also provide the opportunity to estimate cultivation and extraction costs and derive the likely commercial price of the product.
In addition findings will be disseminated as widely as possible.
Second Progress Report
Summary
Period Ending December 2002
Results and Milestones
In the first two years of the Project we have grown indigo-yielding crops in Spain, Italy, Germany, UK and Finland. The crops have been woad (Isatis tinctoria), Chinese woad (Isatis indigotica), and polygonum (Polygonum tinctorium). Using the key yield parameters of leaf weight and indigo yield per unit leaf weight, we have compared genetic lines from a variety of sources; analysed the relationship between indigo yield and the main environmental parameters of air temperature, global radiation, day-length, rainfall, and photosynthetically active radiation (PAR). We found that woad grows well in all the climatic zones examined; while polygonum is more suited to Central Europe and the Mediterranean. To maximise economic yield for both crops, we have identified for each climatic zone the optimum times for sowing, and the optimum times for harvest (up to four harvests per year are possible). Other agronomic factors investigated over the two years are: nitrogen fertiliser rates, irrigation requirements, row spacing, and herbicide treatmentsTraditional breeding methods are being used to select for high-yielding strains of woad, making selective crosses; identifying selection criteria; and analysing pollination patterns.
A reliable technique for measuring these indigo precursors has been developed (paper submitted for publication). The Project has critically examined the existing methods of determining indigo and now accumulated a wealth of information on their application and limitations.
The factors that determine seed production such as vernalisation and day-length have been identified, and sufficient seed can now be produced within the Project for foreseeable future crops.
Mobile farm-based extraction machinery is being developed to maximise the economic recovery of indigo from the crops. Improvements to the design were made in the light of the experience gained in the first year, principally to improve the mobility and throughput. In the second year a prototype visited all five Partner countries to carry out trial extractions. Process parameters have been developed and optimised for woad. Indigo from polygonum has been obtained with traditional extraction systems. For both crops work has been done on the potential use of the plant residues.
The goal of increasing the purity of the indigo to 90% has been approached in two ways. Analysis of indigo being produced in the Project has identified the source of the major contaminant; process modifications based on this knowledge have already reduced impurities. In addition laboratory modelling of indigo formation from indoxyl precursor, has allowed us to follow the actual production of indigo particles quantitatively, and study the interaction of indigo with the impurities.
Indigo produced in the Project has been used in test dyeing to DIN standards with natural fabrics. The results compared well with the synthetic product and commercial sources of natural indigo. In line with the value of our indigo as a natural, sustainable product we have been identifying natural auxiliary agents for dyeing, and we are investigating a novel biological-based means of reducing indigo.
In the second year of the Project we initiated the production of an Environmental Impact Assessment (EIA) of the whole process of natural indigo production, taking note of Council Directives 85/337/EEC and 97/11/EC.
Benefits and Beneficiaries
The progress made in the first two years of the Spindigo Project means that collectively Project Partners can now claim to have a greater concentration of expertise on natural indigo production than can be found anywhere else in the world.
Our commercial Partners' interaction with the textile industry suggests that there is a growing market attracted by the use of natural products, both fabrics and dyes. The Project is in harmony with this development, improving the natural product and identifying what makes it distinctive from the synthetic product; what we like to call refining and defining Nature's fingerprint.
In Year I of the Project our natural indigo was used by a leading London-based fashion house for designs launched at London Fashion Week. This success was followed up in Year 2, when Karada, a leading Italian fashion house used our indigo in a new line (Corpo Nove), of natural fibre men's jackets, launched at Pitti, the major Industry Fair in Florence. At the end of Year 2, a leading Italian knitwear designer, Mely's Maglieria, chose our indigo for their new Kos Collection, which again is to be launched at the Florence Pitti show in early 2003.
Future Actions
In the third year of the project we will continue to gain new information on environment-yield relationships, and crop agronomy. When the accumulated data has been analysed we will be able to produce a package of information for growers. We also plan for further rounds of selection and genetic analysis; improvements to the extraction and purification processes; and further test dyeing.
Progress Report Summary
Period Ending September 2001
Objectives
The aim of the project is to introduce indigo-producing crops into the agricultural systems of the EU so that naturally sourced indigo will supply at least 5% of the European indigo market by 2005, and continue to increase market share, to the long-term benefit of European farmers and the environment. The project aims to deliver a result which can be quantified in terms of hectares grown, number of farmers involved, the regions and areas in which a particular indigo crop can be grown, the environmental impact, and the jobs in the rural sector both created and maintained.
To achieve these aims the project has set itself the following scientific and technical objectives:
At present the European indigo market, which is supplied by the synthetic product, amounts to about 8,000 tomes per year. The price of natural indigo varies greatly, but at a benchmark price of 80 Euro per kg the market is worth 640 m Euros per year. The target market share of 5% would have an estimated annual value of at least 32m Euros per year.
Activities
The workplan follows from the objectives as described above. The overall methodology is based on two kinds of workpackage:
The complete chain of technologies, including the direct effect on landscape and biodiversity of the novel crop, will be subjected to an Environmental Impact Assessment (EIA) for the benefit of the farming industry, planners and environment protection agencies'
Work started on all workpackages on schedule. The two main crops examined were woad (Isatis tinctoria) and Polygonum tinctorium. There was a limited amount of seed of Isatis indigotica, and consequently, less work was done with this species. Indigo crops were grown and analysed in all locations. In two regions, Spain and Finland they were completely new crops. Partners who are growing the crops have obtained access to genotype accessions from a wide range of sources. Collaboration has been extensive throughout the project, including the development of an HPLC system for quantifying indigo precursors in the plants, and the use of plant-derived indigo featuring in designs modeled in the London Fashion Week (Partner 5 grew the woad crop; Partners 2 and 6 extracted the indigo; Partners 1 and 2 liased with the designers; Partner 4 supervised the dyeing).
Results
WP 1 Work started quickly on the method for determination of indigo precursors by HPLC-ELSD. Several types of column coupled to different isocratic elution systems have been investigated in order to resolve the peaks derived from different precursors and their derivatives. It was anticipated that a method for quantification of the indigo precursors would be ready for use by March 2002. Once in place, the method will be used to measure both indican and isatan B. In addition a novel photometric method for the determination of both indican and isatan B was developed, and an acceptable agreement was obtained between the two methods.
WP 2 Woad plants have been selected for yield and have been crossed in the first round of breeding. It was found that indigo precursor potential could be affected by both light and a circadian rhythm, so that it was not possible to switch off indigo production. Work using AFLPs has been carried out on 38 genotypes from Isatis tinctoria, I. indigotica and L glauca. Cluster analysis of the AFLP data revealed the complete separation into distinct groups of the genotypes of each species and landrace. These results provide new and useful information about the make-up of the Isatis genome, which has not previously been evaluated. It will be useful in the selection of plant material for variety development and conservation of the gene-pool. It has been shown that woad is predominantly an obligate out-breeder, genetic variability has been analysed by classical breeding methods, seeds sown from selected plants for a new selection cycle, and genetic variablity analysed by molecular methods. DNA has been isolated for genome analysis and use as molecular markers for marker-assisted selection of high-yielding lines.
WP 3 (woad) and WP 4 (Polygonum) Yield analysis, in terms of leaf weight, precursor content and indigo yield has been started and related to the main environmental parameters of air temperature, day-length, global radiation and photosynthetically active radiation. In the UK, Germany, Finland and Italy an exceptionally wet Spring delayed sowing and led to low yields, especially in the UK, where Polygonum failed as a crop. The most extensive analyses of Polygonum were made by Germany and Italy, as these regions appear to be the most suitable for this crop. As part of these WPs a protocol was adopted for a uniform blocking and sampling methodology in order to make the results from the different European regions comparable.
WP5 (woad) and WP6 (Polygonum) The first season's determination of optimum times for sowing and harvesting the two potential crops were carried out. Again the unusually wet and cold Spring delayed sowing. A surprising finding was the relative success of Polygonum in Finland. Depending on the crop and the region the number of harvests possible varied between one and four. The two key measurements were of leaf weight and indigo yield-
WP6 (woad) and WP7 (Polygonum) Seeds were collected from selections that the most promising performance and bulked. Poor weather in the UK limited production, which was successful in the other countries. The requirements for vernalisation, pollination and seed collection were determined.
WP8 A mobile extraction unit was constructed and tested. This was able to extract indigo from the crops on a farm. All the work carried out in year 1 was in relation to the woad crop. The equipment has been specifically designed to be as flexible as possible so that modifications can be carried out in subsequent years to improve performance. The unit now has a throughput capability of 12 t per day, but the modular design allows a doubling to 24 t per day. The trailer-mounted unit has undergone farm-trials in the UK and Germany.
WP 9 Methods whereby indigo-reducing bacteria might be employed in a process to purify the plant-derived indigo extracts have been investigated. The material extracted from the woad leaves was used as substrate, but- it was found that complex supplements need to be added to this medium in order to support bacterial growth and indigo reduction. Moreover it was found that for reliable indigo reduction, control of several growth parameters was required. The upstream process design is being guided by strict requirements of robustness, simplicity and economy, requirements that were being challenged by the findings made in year 1. As a result, alternative modes of purification were explored. The physical properties of indigo allow for the relatively simple process of sublimation. This was tested and found in principle to be effective, but likely to be expensive in energy and capital costs. As an alternative approach to raising the purity of the indigo, the biochemistry of indigo formation is being explored in order to provide a basis for avoiding production of some of the impurities.
WP10 The purity of samples of indigo extracts has been investigated and where sufficient sample was available, a standard method has been developed for dyeing natural materials (wool and linen). The method has been translated into a set of instructions for industrial use. 'The dyed material was tested for resistance to rubbing and for fastness.
WP11 Progress has been delayed by reservations about the use of indigo-reducing bacteria .
WP 12 Dyeing parameters for use with natural indigo have been optimised and progress made in the identification and testing of natural products as auxiliary agents. Good results were obtained when the auxiliaries were tested according to DIN standards.
Discussion
During the year enquiries received have indicated an increasing demand for natural indigo by the clothing, cosmetic and coating industries. This demand cannot yet be met by Europe's farmers. In the context of a continuing crisis in farm incomes, farmers and landowners have also shown a huge interest in the Project. Thus the Projects importance in providing farmers with the technical means of supplying the commercial demand for natural indigo is now greater than ever.
In the first year, crops of woad have been grown in all regions, and crops of Polygonum have been grown in Germany, Italy, Spain and Finland. Crops have been harvested, analysed for indigo yield, and seeds have been collected. For Central Europe, a case has been made for Polygonum being the crop of choice, but further work is needed on its agronomy and processing for indigo. In the other regions, woad is likely to be better adapted climatically, but results from two seasons are required before final conclusions are drawn. Data to identify key environmental factors that significantly affect indigo yield have been collected, but again further data from the next two years is required before firm conclusions can be drawn. Weeds are currently removed by herbicides, but problems could that arise in gaining Official Approval for use of these on a minor crop. It is also possible that environmental and marketing problems could arise through herbicide use.
The methods used for analysis of indigo precursor and indigo itself have advanced to make the Project Partners world leaders in this field, but the knowledge gained has revealed previously hidden complexities that have delayed completion of this work.
Improvement by breeding is by its nature longer-term, and conclusions cannot yet be expected.
The construction of the mobile unit for high-throughput farm-based indigo extraction has been a great achievement for the first year. As a prototype machine it is readily modifiable, and improvements to its energy use and efficiency of extraction are being made for the next season.
Just enough indigo was extracted to provide for small-scale dye tests and semi-commercial dyeing for a high-profile design company. However in retrospect, larger amounts would have been useful for commercialising the natural indigo, and exploiting more effectively the market demand that the first year has demonstrated exists.
Less progress was made was in developing a biotechnology route for purifying the indigo. As an alternative to the bacteria-based process detailed in WP 9, resources are being diverted to a biochemical analysis of indigo formation to eliminate as far as possible the source of impurity. The original aim of developing a technology that will deliver indigo of a minimum of 90% purity remains.
Future action
In general, work in the second and third years of the Project should adhere to the workplan. However, work in the first year has thrown up additional issues which need addressing, but which were not highlighted in the original proposal. The more significant of these new issues are as follows.
In WP I there is a need to establish a protocol for two of the pre-analytical steps.
Also in WP 1, procedures for an HPLC-based methodology for the estimation of isatan B need to be obtained (in the continuing absence of standard samples of isatan B).
Divergence from the original workplan will take place in WP 9, where resources will be diverted to an analysis of how impurities form in the production of indigo during extraction. This will largely involve an analysis of the biochemistry of indigo formation and the formation of by-products. To this end, an assay of indoxyl formation by spectrofluorimetry has already been developed.
In the second year, the extraction process will be re-jigged in the light of the experience of the first year, to improve throughput, efficiency of extraction, and purity. The machine will undergo trials at various Partner locations.
It has been suggested that the "Project needs interfacing with agri policy with respect to objectives as they could be quite influential on future farming practices". As a result the following statement was added to the Technical Annex: "The project aim is an ambitious one: to introduce a significant new crop into European agriculture. As technical progress towards this aim is realised by the project, agricultural policy makers will be approached with a view to developing possible support mechanisms for the farming systems."
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
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