Periodic Reporting for period 1 - MACSSFT (Development of a Simultaneous Saccharification and Fermentation Technology for Valorisation of Ulva spp. from Macroalgal Blooms)
Reporting period: 2021-09-01 to 2024-08-31
Uncontrolled macroalgal blooms have been on the rise owing to the eutrophication of the marine environment caused by anthropomorphic industrial and agricultural activities. The green opportunistic macroalgae Ulva thrives in such conditions accounting for over half of global blooms. Economic and environmental problems associated with these tides include economic losses in aquaculture, death of marine organisms, inhibition of growth of indigenous flora, the release of hydrogen sulphide from the decaying biomass that is toxic to the native flora and fauna, and high cost of removal and disposal of the accumulated biomass. While environmental and economic problems are associated with these blooms, the biomass generated could be utilised beneficially. The biomass predominantly consists of sugars which account for up to 65% of the dry weight. One of the major sugars- Ulvan is a unique heterogeneous sulfated polysaccharide made up of glucuronic acid, iduronic acid, xylose and rhamnose. These monomers can be used to produce a plethora of products such as aromatics, acetone, butanol, ethanol, and 1,2 propanediol and bioplastics. Owing to the complexity of ulvan, availability of enzymes that can completely breakdown this polysaccharide is a major bottleneck towards its utilisation. Complete chemical breakdown is not possible due to the stability of the bonds that hold the monomers together and is often associated with formation of by-products that hamper further processing. Therefore, the overall objective of the project was to develop a suitable process to utilise the polymeric sugars- ulvan and cellulose from the biomass by breaking them down to simple sugars using enzymes and fermenting them to polyhydroxybutyrate (PHB’s), a polymer that is of interest as bio-derived and biodegradable plastic.
Work carried out for the project can broadly be divided into three parts-
1)Post-harvest/pretreatment optimisation of the biomass
This is an essential step to get rid of physical contaminants, stabilise the biomass and make it suitable for breakdown using enzymes. Parameters optimised include number of wash cycles, dewatering methodology, drying temperature, thermal and pressure pretreatment to break down the structure and make the polysaccharides accessible to enzymes. Additionally, the influence of the age of biomass on its carbohydrates was evaluated using simulated experiments. A manuscript describing the outcome of the study is currently under consideration at the European Journal of Phycology.
2)Developing an enzymatic cocktail for polysaccharide breakdown.
This objective addressed a major bottleneck i.e. non-availability of an enzyme cocktail to completely breakdown ulvan, the major sugar in the biomass to simple sugars. Bacteria capable of breaking ulvan into simple sugars were identified from literature, and 4 were successfully procured for further work. The genes for different enzymes involved in ulvan breakdown were cloned from these 4 bacteria and inserted into a well-studied bacteria- E.coli that has tools available to produce enzymes from these genes in large quantities for industrial use. This can be problematic as the genes are foreign, making them difficult to be used correctly. Ulvan needs 5 types on enzymes for conversion to simple sugars. A total of 29 enzyme genes for these 5 types were cloned. Characterisation (study of optimal working conditions and stability) of 7 novel enzymes that were successfully produced in E. coli was undertaken.
E. coli was unable to produce workable enzymes for all genes cloned, particularly enzymes called sulfatases that are essential to remove the sulfate from ulvan. This highlighted the need for an alternative approach and the genetic modification of one of the bacteria that has all required enzymes was initiated. The bacteria produced minimal amount of enzymes for its survival, hence, the aim was to make it produce larger quantities like E. coli. However, the bacteria was a non-model (not well-studied) organism and a collaboration with an expert in genetic modification of non-model organisms was developed. The proof-of-concept for this work was developed during a EMBO funded scientific exchange grant to the collaborators laboratory to get trained and develop skills required to carry out the work. Additionally, studies were undertaken to understand which enzymes were used by the bacteria from all the genes it possesses. The work done has laid the groundwork for an extensive research project, and funding applications are being made to continue the work.
The second major sugar is cellulose. Cellulose has many commercial enzyme cocktails (cellulases) available for its breakdown owing to years of research. A survey of cellulases that have working conditions similar to the ulvan breakdown enzymes was carried to enable a one-pot breakdown of ulvan and cellulose. The only commercial cellulase that had the desired working conditions was to be discontinued therefore a sample could no be obtained. Cellic® CTec3 a readily available cocktail was used where required for the work.
The work done for this objective was disseminated as an oral presentation at the 24th International Seaweed Symposium at Tasmania, Australia in Feb 2023 and a poster at the Young Algaeneers Symposium 2024, at Vannes, France in May 2024. Two manuscripts based on the work are being written for publication. Results of the characterisation of one novel enzyme has been published in the Journal of Applied Phycology (Rodrigues et al., 2023).
3)Development of a novel fermentation strategy to valorise the unique heterogenous hydrolysate of Ulva spp.
The product of choice for fermentation from the sugars from this biomass was Poly(3-hydroxybutyrate), a type of bioplastic. A P(3HB) producing bacteria that is known to metabolise sugars that are the building blocks of ulvan was selected for this study. The potential of the bacteria to grow and accumulate P(3HB) on the unique heterogenous hydrolysate of Ulva spp. was evaluated. Given that all the recombinant enzymes were not functional, the products obtained from the mix were not the simple sugars that are ideal for fermentation. Three attempts were made using varied protocols; however, production of the desired product was not obtained, most likely due to the sulfation of the sugars and the inability of the bacteria to metabolise the sugars in the sulfated form. The fermentation will be reattempted in the future if funding to resolve the current issue is received.
1)Post-harvest/pretreatment optimisation of the biomass
This is an essential step to get rid of physical contaminants, stabilise the biomass and make it suitable for breakdown using enzymes. Parameters optimised include number of wash cycles, dewatering methodology, drying temperature, thermal and pressure pretreatment to break down the structure and make the polysaccharides accessible to enzymes. Additionally, the influence of the age of biomass on its carbohydrates was evaluated using simulated experiments. A manuscript describing the outcome of the study is currently under consideration at the European Journal of Phycology.
2)Developing an enzymatic cocktail for polysaccharide breakdown.
This objective addressed a major bottleneck i.e. non-availability of an enzyme cocktail to completely breakdown ulvan, the major sugar in the biomass to simple sugars. Bacteria capable of breaking ulvan into simple sugars were identified from literature, and 4 were successfully procured for further work. The genes for different enzymes involved in ulvan breakdown were cloned from these 4 bacteria and inserted into a well-studied bacteria- E.coli that has tools available to produce enzymes from these genes in large quantities for industrial use. This can be problematic as the genes are foreign, making them difficult to be used correctly. Ulvan needs 5 types on enzymes for conversion to simple sugars. A total of 29 enzyme genes for these 5 types were cloned. Characterisation (study of optimal working conditions and stability) of 7 novel enzymes that were successfully produced in E. coli was undertaken.
E. coli was unable to produce workable enzymes for all genes cloned, particularly enzymes called sulfatases that are essential to remove the sulfate from ulvan. This highlighted the need for an alternative approach and the genetic modification of one of the bacteria that has all required enzymes was initiated. The bacteria produced minimal amount of enzymes for its survival, hence, the aim was to make it produce larger quantities like E. coli. However, the bacteria was a non-model (not well-studied) organism and a collaboration with an expert in genetic modification of non-model organisms was developed. The proof-of-concept for this work was developed during a EMBO funded scientific exchange grant to the collaborators laboratory to get trained and develop skills required to carry out the work. Additionally, studies were undertaken to understand which enzymes were used by the bacteria from all the genes it possesses. The work done has laid the groundwork for an extensive research project, and funding applications are being made to continue the work.
The second major sugar is cellulose. Cellulose has many commercial enzyme cocktails (cellulases) available for its breakdown owing to years of research. A survey of cellulases that have working conditions similar to the ulvan breakdown enzymes was carried to enable a one-pot breakdown of ulvan and cellulose. The only commercial cellulase that had the desired working conditions was to be discontinued therefore a sample could no be obtained. Cellic® CTec3 a readily available cocktail was used where required for the work.
The work done for this objective was disseminated as an oral presentation at the 24th International Seaweed Symposium at Tasmania, Australia in Feb 2023 and a poster at the Young Algaeneers Symposium 2024, at Vannes, France in May 2024. Two manuscripts based on the work are being written for publication. Results of the characterisation of one novel enzyme has been published in the Journal of Applied Phycology (Rodrigues et al., 2023).
3)Development of a novel fermentation strategy to valorise the unique heterogenous hydrolysate of Ulva spp.
The product of choice for fermentation from the sugars from this biomass was Poly(3-hydroxybutyrate), a type of bioplastic. A P(3HB) producing bacteria that is known to metabolise sugars that are the building blocks of ulvan was selected for this study. The potential of the bacteria to grow and accumulate P(3HB) on the unique heterogenous hydrolysate of Ulva spp. was evaluated. Given that all the recombinant enzymes were not functional, the products obtained from the mix were not the simple sugars that are ideal for fermentation. Three attempts were made using varied protocols; however, production of the desired product was not obtained, most likely due to the sulfation of the sugars and the inability of the bacteria to metabolise the sugars in the sulfated form. The fermentation will be reattempted in the future if funding to resolve the current issue is received.
The work done towards the project has led to the identification and characterisation of several novel enzymes involved in the conversion of ulvan to simple sugars, which has been/will be published and available to researchers working towards the valorisation of ulvan and Ulva spp. biomass. The work has laid the foundation for the genetic modification of non-model marine organisms to produce biotechnologically relevant enzymes in native organisms as opposed to the use of model organisms. It has also led to the optimisation of an effective post-harvest/pre-treatment methodology for enzymatic breakdown of the complex sugars in the biomass obtained due to green tides. The potential impacts that this work could contribute to is the utilisation of huge quantities of biomass waste that is generated due to green tides and the production of bioplastics could address the current negative environmental impacts of traditional plastics.