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FAIR-CT96-1946
Brassica carinata: The outset of a new crop for biomass and industrial non-food oil - CARINATA |
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Contract No: | FAIR-CT96-1946 |
| Date Prepared: | July 2001, May & September 1999, July 1998 | |
| Source: |
Final Report Second Annual Progress Report - December 1998 First Annual Progress Report |
Coordinator's Overview
The project FAIR CT96 1946 started
officially on January 1st, 1997. However, the kick off meeting took
place in March 1997.
There have been several meeting between the
coordinator team and other participants. The annual meeting took place at
Cordoba, 1, 2 of December, 1998.
I consider that the second year of
achievements is well over the project program
In Task 1. Brassica
carinata as a source of non-food raw material for biomass and
bioenergy chain. There were 2 subtasks each with different items.
The
subtask I . I . Lignocellulose utilisation included the biomass storage
condition, for which different piles of B. carinata were made and later
studied for quality and other characteristics. Among the conclusions were:
The non-food use of B. carinata oil was studied either for
biodiesel production or for plastic additives production (Subtask 1.2.).
Two types of B. carinata oil were studied for biodiesel
production. i.e., high erucic and low erucic acid. During the first part
of the studies, it was shown a positive correlation between the proportion
of methyl linoleate in methyl ester and biodiesel oxidation. The low
erucic acid oil B. carinata was significantly worse than normal
low erucic from rape oil. On the contrary the high erucic B. carinata
oil had similar behaviour than rapeseed oil, in regard to this trait.
Different methyl ester from B. carinata oils were studied
along with antioxidant additives. The promising results found with the
high erucic types and the new low erucic types developed may allow to find
the most appropriate B. carinata oil for biodiesel production.
The evaluation of high erucic B. carinata oil for plastic additives has also shown promising results. The work during 1998 has been focused in the study of sulphur andphosphorus content in high erucic lines grown in different places of the Mediterranean basin. From the obtained results some considerations can be done.
These results suggest that the sulphur and phosphorus amount found in the oil of 4 lines grown in 6 locations is not a limitation for the use of B. carinata oil by the plastic additives industry.
Only one line met the additives industrial requirement (BRK121) in
regard to fatty acid composition. It is important to remark that the lipid
profile was stable in all the tested locations, showing in all the cases a
erucic content over the 50% and a ratio of C18:1/C18:2+C18:3 > 1/2.
In the task 2. Adaptation and productivity of B. carinata in
the Mediterranean agriculture, the general objective was to study the
adaptation and productivity of B. carinata, either as a biomass
crop or as an oil crop for various non food industrial uses, under
different Southern EU environmental conditions. This general objective has
been divided in several subtasks.
Seed yield trials:
In the trials made in the south of Spain
and Italy in the two last years, it could be appreciated that the seed
yield of all B. carinata genotypes was much higher than that of
the rapeseed varieties used as checks.
The results obtained in the
Centre and North of Spain during 2 years of testing, suggest that for
these areas we have to find specific genotypes of B. carinata with
a higher tolerance to low temperatures. Similar results were found in
Central Greece.
Biomass production trials:
In all the trials carried out
during 1997 and 1998 in Spain, Greece and Italy, the top genotypes of B.
carinata showed a dry biomass yield higher than the best check of B.
napus. These results suggest that the tested B. carinata
genotypes are more adapted for biomass production in the Mediterranean
basin than the rapeseed ones.
The evaluation of seeding techniques, planting date and low nitrogen input were also included in the study. The general conclusions for rapeseed in regard to planting date and seeding rate are valid.
In the crop rotations, it seems that B. carinata is better than other common alternatives in Northern Spain.
Some studies have also been made concerning the cost of harvest and transport of B. carinata biomass.
In Task 3 Plant breeding and seed production of B. carinata several subtasks and items were studied.
The breeding work (Task 3.1.) was carried out mainly by the coordinator team. Genotypes were selected by traditional breeding for high yield and oil content, among the two oil types. Also, biomass yield was selected in some genotypes. It seems relatively easy to select for high grain potential. Nevertheless, the progress is slower in increasing oil content and lowering glucosinolate content.
In order to reduce glucosinolate in the B. carinata meal, several alternatives and complementary methods have been used by the teams of Koipesol, ENEA and IAS. This included, classical pedigree breeding, seed mutagenesis using gamma irradiation and gametofitic cells mutations and haploid development after mutation.
Promising results are expected both in classical breeding and by mutagenesis. Nevertheless, it has been found that we may need a separate protocol from the one used for B. napus in studying glucosinolate content in B. carinata meal.
The in vitro regeneration of B. carinata from microspores allows to use this technique in a routine way for the breeding of this species.
This has encouraged as to use a combination of haplo-diploid formation from microspores with chemical and U. V. mutagenesis of the microspores as a most reliable method for mutation breeding rather than looking for somaclonal variation as it was suggested when the project was started.
The hybrid vigor exploitation would be possible in some specific combinations as some CMS sources work well in B. carinata. So far no restorer lines have been developed but the composite system developed in rapeseed could work well in B. carinata as some hybrid tested showed a higher yield potential than their parents.
In general the project is well over schedule and some additional task will be made during the third year.
There is no need to change the original schedule as now additional tasks are well within the proposed project.
Results
The results obtained so far include completion of the second year of biomass characterization, the effect of a year of storage and investigation of various preparation systems. Piles of B. carinata left one year uncovered had lower losses than piles of rapeseed kept under the same conditions. The smallest losses of dry matter and energy have been observed when the piles were covered. There are no significant differences on biomass characteristics caused by modifying the nitrogen fertilization doses or the number of plant per square meter. However, there are significant differences in the ash production during combustion of B. carinata Biomass produced in different areas.
Two types of oil from B. carinata have been studied for the production of methyl esters by transesterification of crude oil. One characterized for a high content in erucic acid, the other a low erucic type. The oxidation stability was compared with other vegetable oils such as: rapeseed oil, sunflower oil and high oleic sunflower oil (HOSO). Among the factors affecting the methyl ester oxidation stability, the more important were:
The presence of unsaturated fatty acids badly affects the oxidation stability of biodiesel. Of the two B. carinata oil samples the low erucic B. carinata oil behavior was significantly worse than that of the rapeseed methyl ester. On the contrary, the high erucic B. carinata oil showed the typical behavior of rapeseed methyl ester despite a different lipid profile. Thus, the low erucic B. carinata line studies proves not to be satisfactory. However the high erucic line look promising. Some new low erucic acid B. carinata types with lower concentration in 18:3 fatty acid are under study.
The evaluation of high erucic B. carinata oil for the production of plastic additives has included several studies. The use of B. carinata oil for the production of plastic additives depends basically on the lipid profile (erucic acid content, and ration among 18C fatty acids) and the content of sulfur and phosphorus in the oil as both can poison catalyst and create corrosion problems. Among different high erucic B. carinata oil types with different fatty acid profiles one line was found which could met the additives industrial requirements.
As regard to phosphorus content in the oil, the 4 B. carinata lines had a relatively low content in 4 of 5 locations studied i.e. less than 100 ppm. Regarding to sulfur content in the oil, most lines and locations showed a similar behavior, with relatively low sulfur content, close to 30 ppm. This is promising, as 50 ppm is the maximum sulfur level acceptable by the industry.
The genotype evaluation and the identification of suitable regions for the production of B. carinata has included several field trials in Spain, Italy and Greece both for biomass and seed production. The results so far obtained indicate that the best production area for B. carinata would be zones characterized by semi-arid climates with mild or hot temperatures. In these areas B. carinata yielded significantly more than the best rapeseed cultivars. This is and area which normally does not produce rapeseed in the E.U. The good adaptation of B. carinata to these areas may be due to its good root system and drought tolerance as well as the lack of silique shattering. On the other hand, B. carinata is less suitable to be grown in areas with frosts unless new cultivars with cold tolerance would be selected.
Among the management techniques studied it has been shown that the best yields are obtained when planting the crop during the early autumn. No differences have been found among different seeding rates from 100 to 200 seeds/m2 for seed production. Yet, for biomass production the best yields in some locations have been found with 200 seeds/m2 . No response was found to nitrogen fertilization between 0 and 150 U.F/Ha. This could probably be due to the use by the B. carinata plant of deep soil nitrogen from previous cereal crops. The effect of this species in the crop rotation showed that cereals grown after B. carinata in two locations in northern Spain yielded better that in the precedent crop was a legume. This was unexpected. Nevertheless, when the water supply is not a limiting factor for cereals, a crop with deep root system may favor the soil drainage and the recirculation of some nutrients such as potassium.
The breeding work has continued as expected. The reduction of glucosinotales content is the main target in the breeding efforts. Also the increase of yield and seed oil content has continued in both high and low erucic types.
There has been a reduction of glucosinolate content in the seed and many lines have about 40 nmol/g. yet, lowering this value may be difficult by conventional breeding. For this purpose, a mutagenesis induced by irradiation was conducted last year. M3 seed from individual M2 plants have been collected. Also, a haplo-diploidization protocol has been established. This is as effective as in rapeseed and therefore suitable for systematic breeding of B. carinata. Thus, two different mutagenesis approaches have been used to develop haplo-diploid mutants: UV irradiation and EMS chemical mutagenesis. Derived plans from treated microspores have been obtained.
The hybrid vigor potential among different crosses of B. carinata lines has been studied in two locations. Heterosis is not presented in all the crosses. Yet, there are lines, which showed a good general combining ability. In addition, there were specific crosses with a grater yield both for biomass and seed yield. The development of B. carinata cytoplasmic male sterile has continued with different cytoplasm sources. Some lines are now CMS. Yet, the development of a fertility restorer is not yet completed.
Introduction
This project started officially on January 1st,
1997, with the kick-off meeting taking place in March. However, in order to
avoid loosing a growing season the agronomical trials were started by the
participants, in the fall of 1996, at their own expense. The project has also
been affected by very abnormal climate conditions over all Europe during last
year. However, in spite of such problems the achievements of the first year, as
outlined below on a task by task basis, exceed those of the projected programme
for the first year.
Achievements
Task 1. Brassica carinata as a source of non-food raw material for the biomass and bioenergy chain
Subtask 1.1. The lignocellulose utilisation
1.1.a. Biomass storage conditions Bales of B. carinata have been constructed in Spain and Greece. These will be under monitoring for one year in order to establish their composition changes.
1.1.b. Biomass characterization Analysis of B. carinata biomass established the content of ash, volatile matter, fixed carbon, major elements (C, H, N, and S) and minor and trace elements content. The preliminary results indicate a relatively high sulphur content which could have a negative influence on the combustion process and emissions. The Brassica species, have this negative trait as they translocate large quantities of sulphur from soil to the biomass. Nevertheless, agronomical practices can reduce the sulphur content in the biomass.
Subtask 1.2. The non-food industrial use of B. carinata oil
1.2.a. Biodiesel production from B. carinata oil For the biodiesel studies, only oxidation stability studies for the biodiesel from rapeseed, conventional sunflowers and high oleic sunflower oils have been completed. Since these are the most common sources of biodiesel in Europe the results will be used for comparison with products from B. carinata oil. As a result of basic chromatographic studies from different B. carinata accessions available in the Koipesol's breeding program, one line with low erucic acid content was selected to complete the studies of B. carinata biodiesel. The oil produced by this line, which was multiplied in Spain during the spring of 1997, was send for biodiesel preparation at the end of 1997.
1.2.b. The evaluation of B. carinata high erucic acid oil for the production of plastic additives and other uses The evaluation of high erucic B. carinata oil (HECO) for the production of plastic additives and other uses was made by studying about 600 inbred lines for oil type within the available high erucic lines in Koipesol's breeding program. Most of the HECO lines showed relatively high erucic acid content. Nevertheless, compared with high erucic rapeseed oil (HERO), the HECO showed relatively high content of C18:2, C18:3, C20:3, C22:0, C22:2, C22:3, C24:0 and C24:1. Thus it seems than HECO genotypes have relatively higher levels of polyunsaturated C18 and C24 acids than HERO. Out of the 600 lines, one had a C18:1/(C18:2+C18:3) ratio similar to that of HERO. This line is under multiplication for further studies. The sulphur content in HECO was also studied, showing similar values to the content found in HERO.
Task 2. Adaptation and Productivity of B. carinata in Mediterranean Agriculture
Subtask 2.1. Genotype evaluation and identification of suitable regions for their production
This part of the work programme has included the evaluation of 6 genotypes for biomass production and 11 genotypes for oilseed production. These evaluations were made in replicated trials in Italy, Spain and Greece. Rapeseed cultivars were included as checks. The seed yields (productivity) of several B. carinata genotypes exceeded that of the best rapeseed cultivars in southern Italy and southern Spain. The findings indicated a very good adaptation of the mustard genotypes to dry and temperate areas of the Mediterranean countries. The higher yields could also be due to the lack of shattering in B. carinata genotypes and greater canopy development in the mustard as compared to rapeseed. On the other hand, the greater yield potential has not been observed in Greece or northern Spain. This could be due to the lack of cold tolerance within the B. carinata genotypes tested. However, the total biomass produced in Spain and Greece was greater in B. carinata than in rapeseed even though 1997 was not a favourable year for the production of biomass due to late planting.
Subtask 2.2. Evaluation of seeding techniques and planting date
Such trials were carried out in northern Greece, southern Italy and northern Spain. It was shown that biomass production and grain yield did not change if the seeds were planted at various sowing rates. This reflected the capacity of B. carinata to modulate its vegetative growth to compensate for differences in plant density within the range of sowing densities tried. The most promising planting rate would be around 200 seeds/m2. In contrast, planting date had a significant effect on both biomass production and seed yield. An early autumn planting date was the best one.
Subtask 2.3. Analysis of low inputs system of production
Two trials concerning nitrogen fertilisation were carried out, one in northern Spain and the second in southern Italy. Nitrogen applications were 0; 50; 100 and 150 units per ha (U.FtHa). In Spain no significant differences, in either biomass or grain yield, were found between the various levels of nitrogen applied. In contrast, in the Italian trial, a positive response to nitrogen was found both in seed and biomass yield. The biomass production was lower at 150 U.FtHa than with 100 U.FtHa. Seed yield, however, increased proportional to nitrogen fertilisation. These results suggest that nitrogen response may vary according to the soil type, availability of nitrogen in deep soil and other agricultural and climatic factors.
Subtask 2.4. The evaluation of the role of B. carinata in the crop rotation
It has just started in northern Spain. Other biomass alternative crops have been grown as well as the cereals.
Task 3. Plant Breeding and Seed Production of B. carinata
Subtask 3.1. Selection of Genotypes and their production
3.1.a. Seed production of B. carinata The availability of seeds of several genotypes has been increased. These will be used for various purposes in the second year (1997/98) trials; including the small scale production of oil for use in biodiesel and additives; and further work on biomass production.
3.1.b. The selection of genotypes for biomass production This task has included the screening of 135 advanced inbred lines for this trait. Half a dozen of lines have been selected for further evaluation in future trials.
3.1.c. The selection of genotypes for grain yield This included the evaluation of a selection of the segregating lines for seed yield within the high, intermediate and low erucic acid genotypes. Oil content in the seed was used as a selection criteria. Of the erucic genotypes, about 30% bred true, maintaining a high erucic values. About 25% of the low erucic lines also bred true. Several lines remained segregant for these traits but some low erucic lines bred true for values around 5%.
3.1.d. Selection of B. carinata with low content of glucosinolates in the seed meal
Subtask 3.2. In vitro regeneration and haploidization of B. carinata
It was found possible to produce haploid of B. carinata, in a very efficient way, by microspore culture. Parameters such as bud size as well as another and petal length have been investigated as means of identifying developing flowers with a high proportion of microspores at the right stage of development of the nucleus. The best protocol, was applied to the most promising B. carinata segregant lines in regard to erucic acid and glucosinolate content. About 60% of the lines tested showed high potential for in vitro culture. This encouraged an attempt to obtain mutants by exposing the microspores to UV light or by chemical mutagenesis with ethylmethanesulfonate (EMS).
Subtask 3.3. Hybrid vigor exploitation
3.3.a. Evaluation of hybrid vigor This work included the crosses among eleven inbred lines in a dialelic manner. A total of 107 different hybrid combinations from a total of 3000 emasculated flowers were made. These hybrids will be evaluated in the second and third year of the project.
3.3.b. The development of a B. carinata cytoplasmic male sterile (CMS) and fertility restorer for the CMS lines This activity has followed the projected schedule. Three male sterile cytoplasms from B. napus are under back-crossing to B. carinata. The Ogura INRA hybrid cytoplasm is now in BC4 showing good CMS in B. carinata. The Polima cytoplasm of B. napus has been crossed to B. carinata for further back-crosses. The third cytoplasm source was Ogura, but the B. napus bearing this cytoplasm did not produce seeds when crossed with B. carinata.
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