In WP3 the main aim under the lead of Bioesplora was the screening and selection for appropriate wild-type strains of Ralstonia eutropha and other strains able to produce PHB. Additional it was foreseen to engineer a recombinant E.coli and to compare PHB productivity with the natural strains.
In order to achieve these objectives, the work package started its activities in month 3 with task 3.1 Screening and selection for wild-type Ralstonia eutropha and with task 3.2 in month 4 Synthesis of recombinant Escherichia coli for PHB production. In month 8 activities of task 3.3 screening for best substrate/microbial strain combination started and in month 18 fermentation optimization experiments were implemented.
Regarding task 3.1 the objectives targeted were the Screening and selection of candidate strains able to produce PHB and the screening of soil and water samples to obtain wild-type R. eutropha. This task was finished successfully with deliverable D3.1 in month 19.
Regarding task 3.2 the final objective was to clone PHA involved genes (phaCAB, prpE, prpP) from Ralstonia eutropha to Escherichia coli. This task was finished successfully with deliverable D3.2 in month 30. In conclusion we have produced a recombinant pET24-CAB E. coli which is able to produce PHB when enzymatic hydrolysates are used as broth culture/carbon source. The recombinat pET24-CAB E. coli accumulate PHB up to 8% of the Cell Dry Weight (CDW).
PHB production monitoring
Regarding task 3.3 the final objective was to select the best combination of substrate / microbial strain and first results could be obtained. This task was finished successfully with deliverable D3.3 in month 35. One wild strain has been selected for each of the proposed fermentation substrates:
- Cupridavidus taiwanensis (E6) for PHBs production from the sweet corn enzymatic hydrolysate.
- Cupridavidus necator (M38) for PHBs production from the potato hydrolysate. These strains have been chosen for scaling-up the process.
The PHBs production with the wild strain (E9: Cupridavidus necator) was of 3.87 g/L with the hydrolysate of sweet corn by-product.
The PHBs production with the wild strain (M38: Cupridavidus necator) was of 5.37 g/L with the hydrolysate of potato by-product.
In WP6 one of the main objectives is to explore the genetic and phenotypic variability that nature has created in order to identify and isolate yeast species able to produce succinic acid. In the scope of TRANSBIO we isolated and identified the yeast flora associated with by-products from fruits and vegetables obtained from processing industries in order to set up a bio-database of yeasts to be screened for their capacity to produce succinic acid and other value added compounds.
It is likely that the yeast flora associated with fruit and vegetable processing by-products is most adapted to the biotransformation of compounds that prevail in these kinds of residues.
A total of 692 yeast isolates were obtained, 450 from the fruit by-products and 242 from the vegetables, revealing that the characteristics of the substrate (nutrients, pH, texture) had influence in the abundance and the diversity of yeast species that were recovered. The genera most frequently isolated were Candida, Cryptococcus, Wickerhamomyces, Hanseniaspora, Pichia, Rhodotorula and Torulaspora. Phenotypic screens for lypolytic and proteolytic activity and for the ability to grow on media made from natural hydrolysates were also performed for all strains. In the last years the utilization of “non-conventional” yeast species has been increasing steadily and this microbial bio-bank offers a great potential for screening yeasts to be used in the future for several biotechnological applications.
The phenotypic screening of “non-conventional” yeast species for the ability to produce succinic acid and the validation of the use of the selected yeast strains in small-scale fermentations was carried out. Two different strains were selected, which were successfully tested in controlled bioreactors. Both chosen strains are able, without any fermentation optimization, to produce higher amounts of succinic acid than the reference strain used.
Evaluation of the differences among a collection of S. cerevisiae strains regarding succinic acid production in different nutritional conditions has also been studied. The results of these experiments have been used to select strains for whole genome sequencing and a comparative genomic analysis regarding the succinic acid metabolic pathway is still underway.
In WP7 an optimized fermentation process is being developed to maximize the production of succinic acid from the diversified raw materials processed in TRANSBIO, such as by-products from fruit and vegetable processing industry, using non-conventional yeasts that have been selected in WP6. This activity is processing real raw materials and is providing real fermentation broths to the partners carrying out the downstream processing (WP8) activities. Further, the yeast biomass that is produced is being sent for assessment of its use for the production of biogas (WP12). The work under WP7 is being performed under the lead of Biotrend (Portugal), in cooperation with the partners UMinho (Portugal) and ttz Bremerhaven (Germany).
WP8 is focusing on the establishment of a down-stream-processing procedure for succinic acid from the fermentation broth produced in WP7. The process of reactive liquid-liquid extraction was evaluated. Effective reactive compounds for carboxylic acids as succinic acid are tertiary amines in an organic solvent. TransBIO has found trihexylamin in 1-n-Octanol to be more efficient then par example trioctylamin as described in many publications. The process of reactive liquid-liquid extraction is shown in the figure below. In the first step the succinic acid forms a reversible amine-acid complex at the interphase. In a second step a back extraction compound is added in order to replace the succinic acid and push into a new water phase. Many trails were performed in order to find the optimal temperature, volume of new water phase, concentration of back extraction compound and time. Oleic acid was used as back extraction compound. After back-extraction succinic acid can be recovered from the new water phase by crystallization and used for further applications in WP13.
Schematic process of reactive liquid-liquid extraction established at ttz Bremerhaven
Compared to other systems reactive liquid-liquid extraction is known for high recovery and selectivity as well as low energy but also high chemical input. Therefore further alternatives will be tested.
In WP10, after evaluating two different protocols (FPLC and precipitation with salts) for purifying enzymes obtained from solid state fermented by-products of rice processing, Proteos Biotech characterized the enzymes obtained in the supernatant and determined their activity.
As responsible of WP11, Proteos Biotech identified the most common components in the formulation of classic detergents (for white and color clothes) and carried out a stability test of 4 week with the supernatant of the SSF against the different components of the detergent formulation.
Trying to increase the stability, Proteos Biotech evaluated the enzymes micro-encapsulation, testing microcapsules with different covers (Poly-L-Lysine, Poly-L- Lysine and Polyethylene Glycol, and Poly-L-lysina-Peptina-Poly-L-lysina-Peptina-Poly-L-lysina (LPLPL)) with two detergent bases (color and white).
However, since the microencapsulation technology seems not compatible with standard detergent formulation (due to chelating agents) new stabilization technologies, such as the crosslink enzymes, will be evaluated. Once a proper stabilization method had been determined, Tecnalia will be able to start the semi-scale test and check the enzymes in a real washing machine cycle.
In WP12 the feasibility of the use of (solid) residuals obtained after hydrolysis of agricultural by-products and fermentation processes as an input material for anaerobic digestion (recuperation of energy and nutrients/fertilizer) is investigated.
First of all, the biogas potential of different input products was determined, both before and after hydrolysis of bio-products. All tested by-products (broccoli, cardoon, green bean, lettuce, potato, sweet corn and banana pulp) showed a good potential for use as (co-)substrate in anaerobic digestion. The Figure below shows the comparison of the biogas potential (expressed on dry matter content) for the original sweet corn by-product and the solid residuals after hydrolysis of this by-product for subsequent use of hydrolysate in TRANSBIO fermentation processes. As it can be seen from this figure, there is hardly any difference in biogas potential after hydrolysis of by-products. Similar results were obtained for solid residuals after hydrolysis of potato by-product. These are promising results and indicate that both high-value chemical production can be combined with energetic valorization (biogas) and fertilizer production (digestate).
Evolution of the biogas production of sweet corn samples
Continuous anaerobic digestion trials are currently being conducted on sweet corn and potato residuals after hydrolysis. So far, all parameters indicate that a stable process can be sustained both in mono-digestion or co-digestion of both substrates.
WP14 aims at conducting an environmental and economic impact study of the novel biotechnological approaches. For this purpose, an initial situation analysis was made in order to construct a reference scenario for comparison with the new TRANSBIO products. From this analysis it was concluded that fruit and vegetable wastes are currently mainly used for energy or fertilizer purposes and not necessarily for bio-products. A literature analysis also revealed that traditional PHB, enzymes and succinic acid production induce a fossil fuel saving, but it is a general impression that information on these pathways is still very limited and that the used mass and energy balances depend on a lot of assumptions and projections. In this work package also the first preliminary environmental assessment results are obtained of PHB produced in the TRANSBIO project through cutting, milling, drying, hydrolysis, PHB production and downstream processing. It was concluded that PHB yield and drying (+other energy) requirement are the most determining factors for the environmental sustainability of this production pathway.