Final Report Summary - PHOTO.COMM (Design & Engineering of Photosynthetic Communities for Industrial Cultivation)
The FP7 ITN PHOTO.COMM: Design & Engineering of Photosynthetic Communities for Industrial Cultivation has contributed to training the next-generation of scientists and leaders in this field. In addition, the project has contributed to the transition of photobiotechnology from a niche specialty towards renewable fuel and chemical production with larger scale production of lower value products.
In PHOTO.COMM we have trained 12 ESRs and two ERs. These are six women and eight men originating from 11 different countries distributed across four continents. From the beginning they have all shown very high enthusiasm and dedication for the project, and they displayed a great team-spirit. The ESRs and ERs each had scientific projects that were interlinked, so they fitted together under four interconnected scientific work packages (WPs): WP1 “Synthetic communities: Strain and community evaluation” (identifying key parameters that underpin stable consortia and understanding of what is important for optimal productivity); WP2 “Systems analysis” (Transcriptome and metabolic modelling based on transcriptome profiling and bioinformatic analysis of cyanobacteria); WP3 “Strain improvements” (Cyanobacterial and algal species will be engineered to improve their robustness and suitability for bioreactors); and WP4 “Industrial cultivation: Up-scaling and evaluation” (Testing the strains provided by WP3 and applying a variety of strain improvement and strain selection methods to test the stains for improved oil content and improved characteristics towards environmental stresses).
In WP1 we have characterized known cultures (from the natural environment and industrial partners) in terms of the algal and cyanobacterial species and their associated bacterial partners. Characterizing and understanding microbial communities that form naturally provide information to help design synthetic communities around a photosynthetic strain that is producing a high value product, thereby making it less susceptible to contamination by undesirable bacteria, and/or reducing nutrient inputs.
We have analyzed the bacterial contaminants of an industrial cultivation of a green alga and found that these were dominated by beta- and gamma-proteobacteria and flavobacteria, which had previously been shown to provide a growth benefit to algae. In complementary work we have analyzed publicly available metagenomics data from marine environmental samples demonstrating high titres of bacteriophages that infect cyanobacteria. Many of these viruses encode components of photosystem I suggesting the possibility that these viruses are important in the maintenance and/or turnover of the photosynthetic apparatus in cyanobacterial hosts.
In a second approach we have shed new light on how mutualism between B12-dependent algae and heterotrophic bacteria that provide the vitamin in return for photosynthate might be put to use to support industrial cultivation of microalgae. A bioinformatics pipeline was established that enabled a comprehensive survey of all sequenced eubacteria for their ability to synthesis B12, including the particular variant required by algae. By this means it was possible to show that the majority if not all cyanobacteria make pseudocobalamin, which is not bioavailable to algae, implying that there is separation of the currency of this vital nutrient in the photic zone. In parallel, a specific co-culture system between Lobomonas rostrata and Mesorhizobium loti has been studied in detailed. Firstly, both proteomics and transcriptomics analysis have been carried out to determine key components that are altered in the alga when engaged in mutualism with the bacteria. At the same time the possibility has been explored of introducing another partner, the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, into the co-cultures to form a consortium. The rationale is that this organism might provide a nitrogen-source that can be used by the other members. One approach was to use several genetic manipulation strategies to increase the release of ammonia into the media by down regulating activity of glutamine synthetase (GS).
In WP2 we have identified regulatory elements critical for the high yield production of metabolites and contributed to the improvement of regulatory models by integration of new regulatory elements. Several different technical platforms for transcriptome analysis, especially tiling microarrays, tailored expression microarrays for the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, and Anabaena sp. PCC 7120, RNA-seq and differential RNA-seq expertise, as well as multiple biocomputational analysis tools have been provided. In particular, the role of regulatory small RNAs and their impact on the photosynthetic apparatus and metabolism including acclimation response to specific limiting nutrients was addressed. Novel functions for genes encoding regulatory proteins and proteins involved in the remodelling and degradation of the photosynthetic apparatus and in modulating electron transport have been identified. Likewise, the role of the non-coding small RNA IsaR1 and another sRNA, the Photomixotrophic growth RNA 1, PmgR1, in the regulation of the acclimation has been elucidated. A novel microarray design that allowed the direct hybridization of labelled total RNA, without reverse transcription or any further enzymatic manipulation has been developed. In addition, we have discovered that many proteins in Synechocystis 6803 involved in absorption of light and photosynthetic reactions are modified via phosphorylation. Thus, a novel level of regulation based on protein phosphorylation could be considered in designing cyanobacterial strains for industrial applications.
In WP3 we have manipulated of key systems in photosynthetic organisms involved in light harvesting, CO2 fixation, nutrient assimilation, and cell wall lysis. Engineered species included the cyanobacterium Synechocystis 6803 and the green alga Chlamydomonas reinhardtii. A major goal has been to use excess photosynthetic electrons and to improve parameters of photosynthesis. This has been achieved by deletion and overexpression of proteins involved in alternative electron transfer pathways in Synechocystis 6803, namely four flavodiiron proteins (Flv1-4), three NADH dehydrogenases (NdbA-C) and the PGR5 (proton gradient regulator 5). Overexpression of Flv2/Flv4 produced the strain that is more resistant to photoinhibition, with reduced production of singlet oxygen and a more oxidized state of the plastoquinone pool in high light conditions. It was found that photosynthesis seems to be limited by the efficiency of native electron sinks, CO2 fixation and carbon assimilation mechanisms. Photosynthetic electrons were dedicated to plant P450 via genetic fusions to ferredoxin.
Several recombinant cyanobacterial and algal strains were created that utilized plant cytochrome P450 enzymes and diterpene synthases. Heterologous enzymes were expressed in active forms, correctly located in cellular compartments and produced desirable products, dhurrin and cis-abienol, respectively. Some strains were grown in bioreactors provided by the industrial partners to corroborate production of these products at the larger scale.
In WP4 we applied concepts developed in the previous work packages (WP1-3) in pilot and pre-industrial settings where “real world” relevance is increased. Participating partners developed at least 5 improved strains of microalgae (GMO or natural mutants) to be tested. These strains were tested at pilot scale at A4F, NovaGreen and AlgaeBiotech industrial partners, allowing students to learn and develop standardized methods for culturing algae. Several PBR systems and configurations were used for the culturing of at least 8 strains of microalgae (5 mutants and 3 WTs as controls). These trials allowed hands-on training of the fellows in scale-up, the development of procedures for pilot and pre-industrial culturing and testing the concepts proven in the laboratory. These tests also highlighted the advantages and weaknesses of each strain in outdoor natural environment conditions. Of particular relevance are the reported successfully cultivation of Phaedactylum tricornutum strains in V-bags, and that they were able to withstand non-axenic culturing conditions. Also a commercial strain of Chlorella vulgaris was cultured mixotrophically in pilot (1m3) and industrial scales (100m3) while maintaining culture longevity and quality. GMO strains of C. reinhardtii were also cultivated outdoors in mixotrophic conditions while maintaining the production of heterologous proteins throughout the course of the experiment. Some observations made in the lab were not translated to the pilot scale. Most notably increased PUFA production was not maintained once the culture was done at pilot-scale suggesting that improvements in CO2 supply should be adopted. Also, consortium tests were unsuccessful demonstrating that a consortium that is well established in the lab can be challenging to translate to outdoors conditions where many other contaminants are also present.
Contact Information:
Project Coordinator Prof. Poul Erik Jensen, University of Copenhagen, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
Email: peje@plen.ku.dk, Phone: (+45) 61 34 46 37
Project website: www.photocomm.ku.dk
In PHOTO.COMM we have trained 12 ESRs and two ERs. These are six women and eight men originating from 11 different countries distributed across four continents. From the beginning they have all shown very high enthusiasm and dedication for the project, and they displayed a great team-spirit. The ESRs and ERs each had scientific projects that were interlinked, so they fitted together under four interconnected scientific work packages (WPs): WP1 “Synthetic communities: Strain and community evaluation” (identifying key parameters that underpin stable consortia and understanding of what is important for optimal productivity); WP2 “Systems analysis” (Transcriptome and metabolic modelling based on transcriptome profiling and bioinformatic analysis of cyanobacteria); WP3 “Strain improvements” (Cyanobacterial and algal species will be engineered to improve their robustness and suitability for bioreactors); and WP4 “Industrial cultivation: Up-scaling and evaluation” (Testing the strains provided by WP3 and applying a variety of strain improvement and strain selection methods to test the stains for improved oil content and improved characteristics towards environmental stresses).
In WP1 we have characterized known cultures (from the natural environment and industrial partners) in terms of the algal and cyanobacterial species and their associated bacterial partners. Characterizing and understanding microbial communities that form naturally provide information to help design synthetic communities around a photosynthetic strain that is producing a high value product, thereby making it less susceptible to contamination by undesirable bacteria, and/or reducing nutrient inputs.
We have analyzed the bacterial contaminants of an industrial cultivation of a green alga and found that these were dominated by beta- and gamma-proteobacteria and flavobacteria, which had previously been shown to provide a growth benefit to algae. In complementary work we have analyzed publicly available metagenomics data from marine environmental samples demonstrating high titres of bacteriophages that infect cyanobacteria. Many of these viruses encode components of photosystem I suggesting the possibility that these viruses are important in the maintenance and/or turnover of the photosynthetic apparatus in cyanobacterial hosts.
In a second approach we have shed new light on how mutualism between B12-dependent algae and heterotrophic bacteria that provide the vitamin in return for photosynthate might be put to use to support industrial cultivation of microalgae. A bioinformatics pipeline was established that enabled a comprehensive survey of all sequenced eubacteria for their ability to synthesis B12, including the particular variant required by algae. By this means it was possible to show that the majority if not all cyanobacteria make pseudocobalamin, which is not bioavailable to algae, implying that there is separation of the currency of this vital nutrient in the photic zone. In parallel, a specific co-culture system between Lobomonas rostrata and Mesorhizobium loti has been studied in detailed. Firstly, both proteomics and transcriptomics analysis have been carried out to determine key components that are altered in the alga when engaged in mutualism with the bacteria. At the same time the possibility has been explored of introducing another partner, the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, into the co-cultures to form a consortium. The rationale is that this organism might provide a nitrogen-source that can be used by the other members. One approach was to use several genetic manipulation strategies to increase the release of ammonia into the media by down regulating activity of glutamine synthetase (GS).
In WP2 we have identified regulatory elements critical for the high yield production of metabolites and contributed to the improvement of regulatory models by integration of new regulatory elements. Several different technical platforms for transcriptome analysis, especially tiling microarrays, tailored expression microarrays for the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, and Anabaena sp. PCC 7120, RNA-seq and differential RNA-seq expertise, as well as multiple biocomputational analysis tools have been provided. In particular, the role of regulatory small RNAs and their impact on the photosynthetic apparatus and metabolism including acclimation response to specific limiting nutrients was addressed. Novel functions for genes encoding regulatory proteins and proteins involved in the remodelling and degradation of the photosynthetic apparatus and in modulating electron transport have been identified. Likewise, the role of the non-coding small RNA IsaR1 and another sRNA, the Photomixotrophic growth RNA 1, PmgR1, in the regulation of the acclimation has been elucidated. A novel microarray design that allowed the direct hybridization of labelled total RNA, without reverse transcription or any further enzymatic manipulation has been developed. In addition, we have discovered that many proteins in Synechocystis 6803 involved in absorption of light and photosynthetic reactions are modified via phosphorylation. Thus, a novel level of regulation based on protein phosphorylation could be considered in designing cyanobacterial strains for industrial applications.
In WP3 we have manipulated of key systems in photosynthetic organisms involved in light harvesting, CO2 fixation, nutrient assimilation, and cell wall lysis. Engineered species included the cyanobacterium Synechocystis 6803 and the green alga Chlamydomonas reinhardtii. A major goal has been to use excess photosynthetic electrons and to improve parameters of photosynthesis. This has been achieved by deletion and overexpression of proteins involved in alternative electron transfer pathways in Synechocystis 6803, namely four flavodiiron proteins (Flv1-4), three NADH dehydrogenases (NdbA-C) and the PGR5 (proton gradient regulator 5). Overexpression of Flv2/Flv4 produced the strain that is more resistant to photoinhibition, with reduced production of singlet oxygen and a more oxidized state of the plastoquinone pool in high light conditions. It was found that photosynthesis seems to be limited by the efficiency of native electron sinks, CO2 fixation and carbon assimilation mechanisms. Photosynthetic electrons were dedicated to plant P450 via genetic fusions to ferredoxin.
Several recombinant cyanobacterial and algal strains were created that utilized plant cytochrome P450 enzymes and diterpene synthases. Heterologous enzymes were expressed in active forms, correctly located in cellular compartments and produced desirable products, dhurrin and cis-abienol, respectively. Some strains were grown in bioreactors provided by the industrial partners to corroborate production of these products at the larger scale.
In WP4 we applied concepts developed in the previous work packages (WP1-3) in pilot and pre-industrial settings where “real world” relevance is increased. Participating partners developed at least 5 improved strains of microalgae (GMO or natural mutants) to be tested. These strains were tested at pilot scale at A4F, NovaGreen and AlgaeBiotech industrial partners, allowing students to learn and develop standardized methods for culturing algae. Several PBR systems and configurations were used for the culturing of at least 8 strains of microalgae (5 mutants and 3 WTs as controls). These trials allowed hands-on training of the fellows in scale-up, the development of procedures for pilot and pre-industrial culturing and testing the concepts proven in the laboratory. These tests also highlighted the advantages and weaknesses of each strain in outdoor natural environment conditions. Of particular relevance are the reported successfully cultivation of Phaedactylum tricornutum strains in V-bags, and that they were able to withstand non-axenic culturing conditions. Also a commercial strain of Chlorella vulgaris was cultured mixotrophically in pilot (1m3) and industrial scales (100m3) while maintaining culture longevity and quality. GMO strains of C. reinhardtii were also cultivated outdoors in mixotrophic conditions while maintaining the production of heterologous proteins throughout the course of the experiment. Some observations made in the lab were not translated to the pilot scale. Most notably increased PUFA production was not maintained once the culture was done at pilot-scale suggesting that improvements in CO2 supply should be adopted. Also, consortium tests were unsuccessful demonstrating that a consortium that is well established in the lab can be challenging to translate to outdoors conditions where many other contaminants are also present.
Contact Information:
Project Coordinator Prof. Poul Erik Jensen, University of Copenhagen, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
Email: peje@plen.ku.dk, Phone: (+45) 61 34 46 37
Project website: www.photocomm.ku.dk