Academic Centre
Project title: Sustainable Microbial Valorisation of Waste Lipids into Biosurfactants (Waste2Surf)
Project No. 1.1.1.1/19/A/047
Source of funding: European Regional Development Fund Specific Objective 1.1.1 “Improve research and innovation capacity and the ability of Latvian research institutions to attract external funding, by investing in human capital and infrastructure” 1.1.1.1. measure “Support for applied research”. Project application selection round No.3.
Project partners: JSC “Biotehniskais Centrs”
Project period: 01.10.2020. – 30.09.2023. (36 months)
Project costs: 647 877 EUR (374 472.90 EUR from ERDF funding)
Project Leader: Senior researcher, Dr.sc.ing. Elina Dace, elina.dace@lu.lv
Objective: to establish a process design workflow for biotechnological production including long-term sustainability modelling, microbial strain design and bioprocess development for sustainable bioconversion of industrial biowaste – waste lipids (used cooking oil, animal fats and others) collected from food production and catering industry into highly valuable biosurfactants.
Summary: Production of high volume valuables from high volume waste is where knowledge-based bioeconomy approach has its greatest potential. The global generation of organic waste is dramatically increasing each year. Waste fats, oils and grease (W-FOG) from food production and catering industry have become a major stream of organic waste in urban areas. According to the EU estimations, each person generates on average 8 litres of used cooking oil per year. The worldwide generation of used cooking oil is about 29 million tons per year. Meanwhile, disposal of W-FOG is challenging, and utilization is limited to bioenergy production. The Waste2Surf project proposes a novel utilization route for W-FOG, i.e. as a low-cost feedstock for production of biosurfactants (BS) – an alternative to non-biodegradable synthetic surfactants synthesized from petroleum, a non-renewable source, through chemical synthesis routes that can be environmentally hazardous.
Surfactants are one of the most important bulk chemicals that are used in almost every product of human daily life – cleaning products, cosmetics, food, pharmaceuticals, etc. In 2024, the global surfactant market is expected to exceed 41 billion euro. The main advantages of BS include their renewable origin, biodegradability, low toxicity, better foaming properties, and stable activity at a wide range of conditions. Considering their advantages BS have a huge market potential, especially when produced from waste.
The project will address the Smart Specialization Strategy’s (RIS3) priority area Knowledge intensive bio-economy, and several target groups will exploit the project’s results.
The main activities of the project will include the development of a novel process design workflow by integrating model-based metabolic engineering of BS producing yeast strains, waste-to-product bioprocess development, and long-term life cycle sustainability assessment of waste-to-product-to-market system. A list of model-based waste bioconversion designs will be delivered ranked according to an integrated criterion considering environmental sustainability, business parameters and social effects. Finally, integration of unconventional separation and aeration techniques into automated bioreactor system will be designed. The bioreactor will be equipped with an advanced model-predictive fermentation control system. As a result, a cost efficient and sustainable system for BS production from W-FOG will be delivered as a new technology prototype (TRL4).
The lab-scale BS’ production will be implemented together with the industrial partner – experienced producer of bioreactors, JSC “Biotehniskais centrs”, established in 1996.
Project report on the tasks completed in the period from 01.10.2020. till 31.12.2020.
During the reporting period, the implementation of the project was started with an in-depth analysis of the scientific literature on the use of yeasts for the production of bio-surfactants from primary and used cooking oils and fats. Based on the literature analysis, five yeast strains have been selected for the production of bio-surfactants and work has begun on characterizing their genotypes. In parallel, work has begun on the development of genome-scale stoichiometric models – a set of genome-scale stoichiometric models has been identified for organisms that produce sophorolipids and mannosileritritollipids. Some of the biochemical pathways producing the target products have been identified or implemented in the models. Work has begun on the development of a laboratory experimental methodology for the determination of biosurfactants, as well as several experiments for the extraction of biosurfactants from waste oils and fats. Two standard methods for the total activity of bio-surfactants (CTAB, oil displacement method) have been introduced. The methods have been tested on both, reference substances and extracts obtained from oil fermentations by yeast.
During the reporting period, the development of specifications for the necessary materials and auxiliary equipment for the fermentation of biosurfactants was started, as well as the identification of critical process parameters and relevant measurement principles and the list of key process model parameters on which the mathematical model development will be based. The first attempt of the fermentation process in a laboratory bioreactor with a strain of S. bombicola was also carried out.
During the reporting period, a team of researchers began work on a review article on the use of yeasts for the production of biosurfactants from edible oils and fats and an assessment of the environmental impact of the production process. A number of communication activities were carried out, as well as three project team meetings to discuss the overall progress of the project, the risks that may hinder the achievement of the project results, and actions to mitigate these risks.
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
Scientific leader: Egils Stalidzāns, e-mail: egils.stalidzans@lu.lv
Administrative manager: Agnese Kukela, e-mail: agnese.kukela@lu.lv
05.01.2021.
Project report on the tasks completed in the period from 01.01.2021. till 31.03.2021.
During the reporting period, work on the project continued with an in-depth analysis of the scientific literature on the use of yeasts for the production of bio-surfactants from primary and used cooking oils and fats. Based on the literature analysis, a series of laboratory experiments was conducted to test the production of bio-surfactants in the presence of various carbon and nitrogen sources. Various analytical methods for quantifying bio-surfactants on site were continued to be sought and adapted directly in the culture solution (medium), reducing the number of extraction steps as far as possible. The use of potassium iodide, stalagmometry and Victoria Pale Blue methods were tested for quantifying bio-surfactants. In parallel, work on the development of genome-scale stoichiometric models was continued. The modelling focused on testing and applying Yarrowia lipolytica genome-scale model to simulate the production of bio-surfactants by adding the missing metabolic pathways that show in detail the uptake of rapeseed oil components in the microorganism. Model validation was initiated with the published experimental data.
Also, during the reporting period, the development of specifications for the necessary materials and equipment for fermentation of bio-surfactants was continued. Parameters for separating membranes of oxygen contactor and bio-surfactants were identified. Based on the information collected from scientific literature, a market study was launched aimed at identifying potential suppliers of equipment elements. In addition, two fermentation experiments with Starmerella bombicola were carried out. One of the experiments succeeded in stimulating the synthesis of sophorolipids.
An in-depth assessment of the impact of waste oil composition and pre-treatment on the production process and final products was carried out. Information on the characteristics and uses of bio-surfactants was also collected in order to identify potential uses of bio-surfactants to be obtained. Data for life cycle inventory analysis have been collected. The work will continue with the life cycle analysis of the fermentation process. The project team continued its work on preparing the review paper of the use of yeasts for the production of bio-surfactants from used cooking oils and fats, the properties and applications of bio-surfactants. Finally, the concept of sustainable metabolic modelling was developed, described and submitted for publication.
05.04.2021.
Project report on the tasks completed in the period from 01.04.2021. till 30.06.2021.
During the reporting period, laboratory and modelling experiments were carried out to characterise the extraction of biosurfactants in lipid-rich fermentation environments. Experiments were continued in the Laboratory of Yeast Nutritional Physiology to adapt analytical methods for quantifying biosurfactats and carbon sources (lipids/glucose) on site directly in the culture solution (medium), minimizing the required treatment. The suitability of the glucose oxidase and antrone-sulphuric acid test for the quantification of biosurfactants was tested, and yeast growth tests were performed in the presence of oils (including waste cooking oils) and additionally added carbon and nitrogen source. The growth of yeast biomass under different conditions (in the presence of fresh/used oil and different sources of nitrogen) and the production of biosurfactants under the given conditions, as well as the chemical composition of raw materials (oils) and biosurfactants produced, were assessed. In the Fermentation Laboratory of the JSC Biotehniskais centrs, a number of experiments focusing on the removal (filtration) of biomass/product during the fermentation process were carried out, as well as biosurfactant washing and enconcentring experiments with two variations of the tangential filtration system (10 and 20 kDa membrane) were carried out. The experiments resulted in the collection of the required experimental data on the performance of the biological system in the production of biosurfactants.
Modelling tasks continued work on the development of genome-scale stoichiometric models, as well as work on the structure of kinetic models. During the reporting period, work was continued on adaptation of the Yarrowia lipolytica genome scale model. Work on the development of the stoichiometric model of Starmerella bombicola was launched on the basis of available sequencing data. In order to improve the quality of initial metabolic reconstructions, a variety of software for analysis of sequencing data is used in the design of models. In parallel, the development of a structure of kinetic models for the manufacture of surfactants was initiated using the models of Starmerella bombicola and Yarrowia lipolytica. A variety of scenarios for which life cycles will be compared in the follow-up study process were developed and evaluated to determine the environmental impact of the biosurfactant production scheme. A detailed structure was established for the basic model providing for a separate environmental impact assessment for medium, yeast fermentation and various fermentation regimes. At the same time, work was launched on collecting and processing data on the social and economic parameters of the management of waste cooking oils.
In cooperation with laboratory and modelling team researchers, work on the preparation of a scientific article on the use of yeasts for the production of biosurfactants from waste cooking oils and fats was completed. The manuscript was submitted to the journal Fermentation (ISSN: 2311-5637, IF (2020) = 3.975).
07.07.2021.
Project report on the tasks completed in the period from 01.07.2021. till 30.09.2021.
During the reporting period, experimental work continued actively both in the Laboratory of Yeast Nutritional Physiology and in the Fermentation Laboratory of the JSC Biotehniskais centrs. Experiments were continued in the Laboratory of Yeast Nutritional Physiology and a method to determine the presence of biosurfactants more efficiently and conveniently in lipid-rich fermentation media was developed. The method uses a combination of several methods, that is specifically adjusted to detect glycolipid biosurfactants. The developed method is described and will be used as a basis for a new scientific publication. The research team has also developed a method for biomass quantification based on the number of cells per mL during the reporting period. The growth of Starmarella bombicola was evaluated using glucose, glycerine and fatty acids as growth substrates. In each of these experiments, the biomass-specific growth rate was determined using the developed biomass quantification method.
The development of the pilot plant in Fermentation Laboratory of the JSC Biotehniskais centrs has continued. The installed tangential filtration module works well and can filter 100 mL/min. As a result of the procurement, several components of the reactor fermentation/extraction system, including peristaltic pumps, a surfactant storage tank, membranes, have been purchased and the assembly of the system has started. During the reporting period, several S. bombicola cultivation/fermentation experiments were carried out to determine the kinetic parameters of the process. Parameters will be used to develop options for softsensor algorithms. The results of the previous experiments were analysed, and it was observed that the foaming of the fermentation medium can be partially (or completely) prevented by maintaining the pH of the medium at low values (<3.5). This, in turn, makes it possible to avoid the use of gas contactors to produce surfactants.
In the modelling sections of the project, work continues with stoichiometric and kinetic models, which used simultaneously improves the reliability of the simulated results. A genome-scale model of S. bombicola is being developed to assess the engineering potential of the organism. Recent genome-scale models for Yarrowia lipolytica are also under consideration. The structures of kinetic models are extended to incorporate different substrate components.
To determine the environmental impact of the bioactive production scheme, additional aspects that may influence the model results were evaluated. The specificities of life cycle modelling of biological processes have been given increased attention. The most appropriate solution is being sought for modelling of fermentation processes. Work has also started on the modelling of the system dynamics of waste cooking oils by developing a conceptual model of the system and a causal loop diagram.
The research work has resulted in two scientific articles prepared and published:
- Liepins, J, Balina, K, Soloha, R, Berzina, I, Lukasa, LK & Dace, E 2021, 'Glycolipid biosurfactant production from waste cooking oils by yeast: Review of substrates, producers and products', Fermentation, vol. 7, no. 3, 136. on the use of yeasts for production of biosurfactants from waste cooking oils and fats.
- Stalidzans, E & Dace, E 2021, 'Sustainable metabolic engineering for sustainability optimisation of industrial biotechnology', Computational and Structural Biotechnology Journal, vol. 19, pp. 4770-4776. on the concept of sustainable metabolic engineering, which allows the sustainability of biotechnological production to be assessed and optimised by deriving it from the metabolic traits of the micro-organism used.
The project activities and results were presented to industry professionals at the COST Action Yeast4Bio: Non-conventional yeasts for the production of bioproducts (CA18229) meeting (29.09.2021, remotely).
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
Scientific leader: Egils Stalidzāns, e-mail: egils.stalidzans@lu.lv
Administrative manager: Agnese Kukela, e-mail: agnese.kukela@lu.lv
05.10.2021.
Project report on the tasks completed in the period from 01.10.2021. till 31.12.2021.
During the reporting period, work on both mathematical modelling of metabolism and modelling of environmental impacts has been ongoing. A system dynamics model is also beginning to take shape, but further work is needed to refine the model and develop potential scenarios. During the reporting period, experimental work continued actively both in the Laboratory of Yeast Nutritional Physiology and in the fermentation laboratory of the project partner JSC “Biotehniskais centrs”.
A prototype of fermentation/extraction system for surfactants was successfully validated in the yeast physiology laboratory with model environments. The specificity of the anthrone and DNA methods (lipids, other surfactants) was tested. A protocol for the application of the methods for the determination of biosurfactant in lipid-rich fermentation media was prepared. Work on the recombinant strains’ method has started: literature analysis is being carried out to select the optimal Starmerella bombicola transformation and recombinant selection methods.
Work on optimisation of the fermentation and extraction system for biosurfactants is ongoing in the fermentation laboratory of the project partner JSC "Biotehniskais centrs". Optimal operating parameters (pressure, flow rate) for the removal of sophorolipids/biomass from the fermentation medium were determined. During the reporting period, several S. bombicola cultivation/fermentation experiments were carried out to determine the dynamics of biosynthesis. Based on these parameters, both – a mathematical model of the process and a softsensor algorithm are being developed. Screening experiments are being carried out with the aim to identify yeast extract constituents that stimulate biosynthesis of sophorolipids in S. bombicola cells. The experimental determination of the metabolic diversion of amino acids in yeast extract to the biosynthetic pathway of biosurfactants was carried out. Experiments are being carried out to determine which amino acids stimulate biosynthesis and at which concentrations.
The modelling teams of the project continue the process of building a genome-scale model of S. bombicola. In collaboration with the yeast physiology laboratory, the selection and combination of the most appropriate models for Yarrowia lipolytica continues. Parameters of the kinetic models are being determined by parameter estimation methods in the COPASI environment, based on published and experimental data generated within the project, to determine the pathway of biosurfactant production. The design of strains based on the synergy of stoichiometric and kinetic models has been initiated by optimising the available models according to the developed prototype solution ranking criteria.
During the reporting period, the basis of a life cycle model of sophorolipids production was developed. The model simulates the environmental impact of the fermentation process and identifies the production steps where environmental impacts occur. The model defines parameters that can be changed to be adapted to the fermentation process. Work has started on the preparation of the life cycle assessment report. During the reporting period, work has started on the development of a system dynamics model and data are being collected to identify the patterns that determine the dynamics of the model.
Two abstracts were submitted to the international scientific conference "Conference for Young Scientists on Biorefinery Technologies and Products", which will take place on 27-29 April: www.btechpro.lv
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
Scientific leader: Egils Stalidzāns, e-mail: egils.stalidzans@lu.lv
Administrative manager: Agnese Kukela, e-mail: agnese.kukela@lu.lv
14.01.2022.
Project report on the tasks completed in the period from 01.01.2022. till 31.03.2022.
During the reporting period, experiments have continued to determine the optimal fermentation environmental conditions to produce sophorolipids by fermentation. The process of production of biological surfactants is becoming more and more concrete. During the reporting period, activities for the development and optimisation of kinetic and stoichiometric models continued. Work on modelling the environmental impact of the process is also continuing and the model is being adapted to match experimental results. An environmental impact assessment report for the baseline scenario has also been prepared.
The Yeast Physiology Laboratory carried out laboratory tests on a variety of yeasts, using both raw cooking oil and waste cooking oil. The tests showed that Starmarella bombicola does not lose its viability, while Yarrowia lipolitica cells die after the fifth day of the experiment. Vitamin auxotrophy of S. bombicola was also determined. The tests showed that the presence of biotin is critical for the growth of S. bombicola. A literature analysis of the sophorolipid production pathway in S. bombicola metabolism is being carried out to allow genetic engineering in the strain. Deletion of the Ura3 gene results in S. bombicola no longer producing uracil. Carbon source consumption was also analysed. It is concluded that glucose, fructose, and glycerol are the most consumed. This is important information in the search for a gene to knock out in the engineering process. An experiment has been launched to knock out the ura3 gene with 5-FOA. By knocking out the ura3 gene, uracil will have to be artificially added, but this will allow to control the process.
In the Fermentation laboratory of the project partner JSC "Biotehniskais centrs", the second prototype fermentation/extraction system for biosurfactants has been built and successfully tested in a fed-batch fermentation process. The optimum operating parameters (flow rate) for the removal of sophorolipids/biomass from the fermentation medium were determined. During the reporting period, several S. bombicola cultivation/fermentation experiments were carried out to determine the dynamics of the synthesis of biosurfactants. Based on these parameters, a mathematical model of the process was developed and a softsensor algorithm is under development. Screening experiments are being carried out to identify the yeast extract components that stimulate soforolipid biosynthesis in S. bombicola cells. The metabolic diversion of amino acids in yeast extract towards the biosynthetic pathway of biosurfactants was experimentally determined. Experiments are being carried out to determine which amino acids stimulate biosynthesis and at which concentrations. Fermentation of S. bombicola using different synthetic/complex media compositions was carried out. Syntheses of bioactive substances were initiated, and the kinetic parameters of the processes were evaluated. Based on the kinetic parameters, a process control algorithm was developed and refined.
In the modelling tasks of the project, an initial version of the S. bombicola model has been established and will be refined in the light of experimental results. The genome-wide adaptation of the two Y. lipolytica surfactant-producing strains to the project is ongoing. Models of other surfactant-producing organisms are also being analysed with a view to their inclusion in the optimisation workflow. The metabolic pathways involved in surfactant production in Y. lipolytica are analysed in detail. The resulting steady-states of metabolism are transferred to genome-scale stoichiometric models to determine the feasibility of pathways taking into account biomass production. The framework of the complex optimization algorithm in the COBRA toolbox has been developed, which allows determining the steady-state fluxes of the organism while it is being optimized. The resulting steady-state fluxes are tested in kinetic models.
During the reporting period, a study on the main flows of edible oils was carried out and an initial model was developed. Optimisation of the structure of the environmental impact assessment model was performed. The model identifies the phases with the highest environmental impacts and seeks solutions/scenarios for mitigation in cooperation with project partners. Environmental impact indicators have been selected. It is considered to use them to obtain single indicator using normalisation and weighting techniques.
During the reporting period, several communication activities were carried out. ". The results of the project were presented at the 80th International Scientific Conference of the University of Latvia. Two additional abstracts for participation in international scientific conferences were submitted. The project was featured on the Latvian Radio programme "Zināmais Nezināmajā" (Known in the Unknown). In addition, participation in a national event for professionals and the general public was carried out by giving a presentation at the Vidzeme Innovation Week 2022 event. During the reporting period, an information webinar for professionals and the general public on the project results and future activities was also organised, as well as an international training workshop Biomodelling Spring 2022. Information about the project at the partner’s website: www.bioreactors.net/wastetosurf
18.04.2022.
Project report on the tasks completed in the period from 01.04.2022. till 30.06.2022.
During the reporting period of the project, synergies between kinetic and stoichiometric models have been developed through optimisation experiments. Preliminary results of the environmental impact assessment have been presented at two conferences and the system dynamics model of the waste cooking oil is becoming more concrete. Experimental work in the laboratory continued with experiments on Starmarella bombicola and the development of a mutant of the yeast Yarrowia lipolitica. Work is also continuing on the characterisation of the operating parameters of the extraction/downstreaming process. Empirical mathematical relationships have been established which will allow future modelling and/or designing of similar systems for different scales of sophorolipid production.
Laboratory tests were carried out in the Yeast Physiology Laboratory to determine growth parameters (glucose consumption, growth rate, mannitol accumulation, biomass components) of S. bombicola in batch fermentation tests with glucose. The experiment to eliminate the ura3 gene with 5-FOA was completed. By eliminating the ura3 gene, uracil needs be added, but this facilitates manipulation of the organism functioning. The ura3 mutant of Yarrowia lipolytica that was created has been deposited in the microorganism collection at the Institute of Microbiology and Biotechnology. Preparations (first tests) have started to use this strain as a basic chassis for genetic engineering.
In the project sections devoted to kinetic and stoichiometric metabolic modelling, metabolic pathways of the genome-wide models are built. They may operate in parallel to the pathways included in the kinetic models. The analysis of the variability of these pathway fluxes allows to determine their potential impact on the process under study at specific values of biomass growth. Different types of constraints are applied in the kinetic models, which improve the likelihood of applicability of the developed solutions in biological experiments. Genome-wide stoichiometric models are optimised using different combinations of deletions and insertions, maximising the total carbon flux to the products, which minimises the release of carbon into by-product compounds. The convergence rate during optimisation is evaluated. Work is in progress on the first strain designs.
The environmental impact assessment of the baseline model has been completed during the reporting period and environmental hotspots have been identified. Several scenarios to address the hotspots have been developed and presented as suggestions at an international conference. In consultation with an external expert, the points requiring further attention in LCA model have been identified: 1) Functional unit, 2) Use scenario, 3) Waste scenario. The literature analysis and the expert suggestions have been used to address the existing uncertainties and gaps. In the system dynamics modelling activity, causal loops have been identified and characterised. Data analysis is carried out to describe the loops by mathematical formulae. The construction of a yeast metabolite database to describe the environmental impact of each metabolite entering and leaving the yeast cell has started.
Project partners JSC "Biotehniskais centrs" worked on the characterisation of the operating parameters of the extraction/downstreaming stage of sophorolipid production process. Empirical mathematical relationships were established which will allow future modelling and/or design of similar systems for different scales of sophorolipid production processes. Cultivation/fermentation experiments also continued during the reporting period. These experiments included the accumulation of relevant data and the development of softsensor algorithms for testing. Since the start of the project, due to the constraints imposed by Covid-19, the experiments foreseen under the activity have been delayed and the activity is expected to be completed by 31 December 2022.
Screening experiments using different fermentation media compositions (varying concentrations of both amino acids and other organic/inorganic components) were continued to identify yeast extract components that stimulate sophorolipid biosynthesis in S. bombicola cells. Experimental and theoretical information is combined to look for correlations.
During the reporting period, several communication activities were carried out including three presentations on the project results at two international scientific conferences.
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
12.07.2022.
Project report on the tasks completed in the period from 01.07.2022. till 30.09.2022.
With the finishing of previous project reporting period, the work on the life cycle assessment has been completed and the results are summarised in an LCA report. In the meantime, analysis on sophorolipid pathway in various organisms is still ongoing, with plasmid constructs for heterologous expression of the synthesis pathway being generated. Studies to characterise the growth phenotype of S. bombicola are also ongoing. Experiments with model medium (sophorolipid solutions) on the established filtration system are being carried out in the laboratories of the collaborating partners JSC "Biotehniskais centrs". A plan for the filtration experiments has been established and is being implemented. The data obtained will be further used to model the performance of the filtration process. During the reporting period, the optimal composition of the culture medium in which to efficiently stimulate the synthesis of sophorolipids was also established. Experiments with model media (sophorolipid solutions) are being carried out on the developed filtration system. Based on the above, experimental data (for both fermentation and filtration processes) are being actively accumulated, which will further allow the adaptation/development of an efficient control system.
Additional literature data are being collected to develop kinetic models for biosurfactant production and to optimise it in order to define more accurate model parameters. Possible differences between the pathway parameters of Y. lipolytica (after engineering) and S. bombicola are also sought. The types of reaction kinetics equations are examined. Evolutionary algorithms are being trained to design strains for simultaneous searches for deletions and insertions to improve speed and convergence properties. The implementation of growth-coupled production methods in optimisation continues. Ten strain designs based on the synergy of stoichiometric and kinetic models have been developed.
Final refinements have been made to the life cycle assessment model. The inaccuracies that had arisen during the previous project period by assigning two different functions to the model have been eliminated. The results of the life cycle assessment have been prepared for presentation at an international scientific conference. The report on the results of the life cycle analysis has been finalised. Work on the development of the system dynamics model also continued during the reporting period. The model is divided into two main structures: the waste generation system and the biosurfactant market. The interlinks between the two models are being explored. Socio-economic indicators affecting the system have been discussed during the reporting period. A literature analysis is carried out to identify the most appropriate indicators to describe the system.
Work on the determination of growth parameters of S. bombicola has been completed in the Yeast Physiology Laboratory. A review of the physiology of the standard strain of S. bombicola has been prepared. A set of Yarrowia lipolytica goldengate plasmids was purchased and deposited. Gene sequences of S. bombicola soforolipid fusion proteins corresponding to the GoldenGate cloning system for Y. lipolytica were generated. Biosurfactants produced by Y. lipolytica have been characterised. Tests with Pseudozyma antarctica have been initiated to test the ability of this micro-organism to convert waste cooking oils.
Cultivation/fermentation experiments are continuing in the laboratories of the project partner JSC "Biotehniskais centrs". Based on the results of these experiments, a softsensor algorithm is being developed. Screening experiments to identify yeast extract components that stimulate sophorolipid biosynthesis in S. bombicola cells were completed during the reporting period. Further experiments will be carried out with an appropriate composition of the fermentation medium in a 5L bioreactor for proof of concept.
Empirical models are being developed to predict the main performance parameters of the sophorolipid extraction system and theoretical scaling is being realised. The work on the report is ongoing. The optimum composition of the medium in which to efficiently stimulate sophorolipid synthesis was established. Based on the results of the cultivation/fermentation experiments carried out, the existing version of the algorithm was further developed. The efficiency and overall functionality of the algorithm is tested with existing experimental data. The development of the optimisation algorithm is under active development. The first versions are being tested on the experimental data.
During the reporting period, several communication activities were carried out, three presentations on the project results were given at three international scientific conferences.
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
27.10.2022.
Project report on the tasks completed in the period from 01.10.2022. till 31.12.2022.
During the reporting period, the Computational Systems Biology Group continued to refine and extend their models to provide the most complete representation of the mechanism of sophorolipid synthesis. Meanwhile, in the Sustainability Modelling Group, work continued to improve the system dynamics model and describe the dynamics with mathematical equations. A publication based on the results of the life cycle analysis is under preparation. Planned experiments on a sophorolipid filtration system have been completed in the laboratories of the partner Bioreactors.net. The data obtained were partially compiled and analysed. Empirical mathematical relationships between filtration rate, effective filtration area, membrane pore size and retentate circulation rate were constructed. These empirical relationships (models) are being validated. In the yeast Physiology Laboratory, constructs for the expression of the Starmarella bombicola sophorolipid synthesis pathway in Yarrowia lipolytica have been developed. Also, experiments with Pseudozyma antarctica, started in the previous reporting period, were continued to test the ability of this micro-organism to convert waste cooking oils. Experiments were also continued with the optimal composition of the fermentation medium found previously. The experimental data are collected and analysed to adapt the process control system. During the reporting period, more attention was paid to mass balances as well as to the identification of process markers able to describe the current state of the bioprocess (synthesis of sophorolipids/biomass growth/formation of other metabolites).
Three kinetic models have been developed to model and optimise the scale kinetic model of the sophorolipid production pathway: two models describing sophorolipid production by S. bombicola on different substrates and one model describing the production of a surfactant precursor in Y. lipolytica. The models have been subjected to TOP optimisation, resulting in the identification of metabolic engineering targets. The development of new strain designs is continued with introduction of environmental and social sustainability criteria, using the OptGene approach as the basis for the search algorithm. The developed projects are tested for their ability to incorporate the product production flows described in the above three kinetic models.
To assess the sustainability of sophorolipid production using waste cooking oil, additional literature data is being collected to improve the existing structures of the system dynamics model. In collaboration with colleagues of the Yeast Physiology Laboratory, an approach is under development to model the conversion of waste oils to biological surfactants, taking into account the relative proportions of substrates. The effect of the glucose-waste oil ratio on the yield of sophorolipids is planned to be used as a co-relation. Based on the literature analysis different scenarios for the multicriteria analysis are being developed. Socio-economic indicators have been identified, from which the most relevant ones will be selected to compare the scenarios developed.
A soft-sensor algorithm for sophorolipids has been developed in the project partner’s Bioreactors.net laboratory. Relevant information (algorithm and description of methodology) was submitted for evaluation. An effective strategy was found to reduce foaming without impairing oxygen mass transfer. The evaluation of the techniques investigated, and the description of the final effective strategy have been submitted for evaluation. Based on the obtained results, a prototype fermentation/extraction system is being developed. The resulting filtration process models are used to evaluate the predicted efficiency and relevant system performance parameters during the prototype development. During the reporting period, the MPC control algorithm was significantly enhanced (integrating efficient mass balance accounting algorithms and testing several process efficiency markers (RQ, OUR, CER) and their combinations). Work on the improvement of the MPC system is ongoing. The latest version of the MPC control optimisation algorithm demonstrated good results and proved its efficiency in bioprocesses to produce sophorolipids. Additional experimental data are being collected to further improve the performance of the algorithm.
Several communication activities were carried out during the reporting period. A poster presentation and an abstract on the project results were presented at the International Scientific Conference "LCA Foods 2022". A general presentation on the project and its results was also given to colleagues from other departments of the University of Latvia.
The project team visited the production site of Happy Fish Ltd, a company producing biosurfactants from oil. During the visit, cooperation opportunities were discussed, and the participants exchanged experience on aspects of the production of biosurfactants.
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
06.02.2023.
Project report on the tasks completed in the period from 01.01.2023. till 31.03.2023.
During the recent phase of the Waste2Surf project, considerable strides were made by our Genome and Sustainability Modelling groups. The Genome Modelling group augmented their focus on microorganism strain modelling by fusing kinetic and stoichiometric models. Simultaneously, the Sustainability Modelling group honed the system dynamics model, seeking optimal ways to integrate its diverse structures. Meanwhile, the Yeast Physiology Laboratory demonstrated their proficiency by successfully cloning heterologous genes into the Yarrowia lipolytica genome following the Golden Gate (GG) protocol. Concurrently, the team at JSC “Biotehniskais Centrs” pressed forward with experiments using an optimally determined growth medium.
We are thrilled to announce that the prototype of the biosurfactant extraction system has been seamlessly integrated into the bioprocess operating system. This allows us to collect vital experimental data from fermentation and separation processes, thereby refining our control of the continuous process. Furthermore, the prototype completion signifies a notable accomplishment in our project activities.
The Metabolic Modelling group, during this reporting period, fortified their application of kinetic and stoichiometric model synergy, leading to higher potential for positive outcomes. Increased success rates were achieved by implementing limitations initially omitted in the model, while details of the stoichiometric model continue to be enhanced.
Our Sustainability Modelling group has now pivoted its focus towards developing a waste cooking oil valorisation module within the system dynamics model. The module focuses on three crucial areas: identifying various waste oil usage pathways like biosurfactants, biodiesel, and biogas; devising an optimal biosurfactant production pathway, considering factors such as glucose to waste oil ratio and maximum biosurfactant yield; and initiating work on the biosurfactant consumption and economic modules.
During the last reporting period, we made considerable strides in research on Multi-Criteria Decision Analysis (MCDA) methods. Our objective was to identify the most apt and relevant MCDA methods in line with our project goals. A series of in-depth discussions and exchanges of ideas took place among working groups to ensure a logical analysis of scenarios. The subsequent plan is to implement the identified MCDA methods to scrutinize data and draw conclusions.
In the Yeast Physiology laboratory, the first successful cloning of heterologous genes in the Y. lipolytica genome has been accomplished per the GG protocol, with active gene expression demonstrated. Our team will now proceed with the cloning of the remaining sophorolipid pathway genes and the preparation of vectors in accordance with the GG protocol. Notably, surfactant by-product potential was explored as an additional additive for Starmerella bombicola biomass production.
Finally, this period marked the completion of the biosurfactant extraction system prototype. The prototype employs a two-step continuous filtration of the fermentation broth, initially separating biosurfactants, and then enhancing their concentration. The creation and validation of the prototype entailed conducting experiments with both simulated and real fermentation broths.
During this period, we accumulated additional experimental data to augment the MPC control algorithm. Concurrently, we tested the algorithm's stability using artificial data.
Information about the project at the partner’s website: https://www.bioreactors.net/wastetosurf
11.05.2023.
Project report on the tasks completed in the period from 01.04.2023. till 30.06.2023.
As the project enters its final phase, more focus is placed on summarizing and analysing results thus concluding some of the project activities. During this reporting period, modelling efforts continued in both the Genome Modelling Group and the Sustainability Modelling Group.
The Metabolic modelling group has developed cell designs that could be considered candidates for laboratory implementation. These designs offer numerous alternative pathways for gene expression, with a significant number of implementation options to be analysed. Some designs focus on producing bioactive compounds using alternative gene-protein-reaction associations. The completion of cell design development marks a significant milestone in this activity.
The Sustainability modelling group has concluded work on the system dynamics model and is now proceeding with multi-criteria analysis to assess the sustainability of sophorolipid production. The model's individual structures have been enhanced to reduce uncertainties. Simulations have captured the system’s state for the years 2020, 2030, and 2050. The system dynamics model, its modules, and various scenarios are detailed in a project report. As the system dynamics activity concludes, the project's final phase will focus on comprehensive multi-criteria analysis to evaluate the sustainability of sophorolipid production. Ongoing work includes the selection of indicators and value determination.
In the Yeast physiology laboratory, heterologous gene cloning has demonstrated the activity of expressed genes. Four out of six genes have been prepared for expression in Y. lipolytica using the GoldenGate protocol. The construction of recombinant cells will continue until the project's end. A method has been adapted for assessing the production of bioactive compounds using Y. lipolytica.
At the partner institution, fermentation experiments using the Model Predictive Control system have been conducted. Data from these experiments have been consolidated for reporting purposes. Testing and refinement of the optimization algorithm are ongoing, focusing on identifying states where the algorithm may not perform effectively. Additional parameters will be incorporated into the system to enhance the recognition of sophorolipid synthesis and biomass growth states.
Throughout the reporting period, several dissemination and communication activities have been implemented, including poster presentations in two international scientific conferences. In addition, a three-day seminar-training course "Biomodelling Spring 2023" was organized to promote and share knowledge on the metabolic modelling methods used in the project.
05.08.2023.
Project report on the tasks completed in the period from 01.07.2023. till 30.09.2023.
During the final period of the project, the main focus was placed on obtaining and summarizing results to conclude the project activities. Final steps of sustainability assessment were conducted using multi-criteria decision analysis (MCDA). Additionally, recombinant strains of yeast Yarrowia lipolytica were developed and described. Final steps of model predictive control (MPC) system optimisation were carried out.
The project team finalised the sustainability assessment for the years 2020, 2030, and 2050 and analysed the results. Future scenarios were calculated by assuming changes in electricity mix in future scenarios and by calculating future prices of oil substrate. Two reports on the sustainability assessment results have been prepared. One report includes step-by-step selection on sustainability criteria while other report describes MCDA methodological choices and results.
The project team has completed the construction of the GoldenGate construct in the Yarrowia lipolytica yeast strain, and recombinant strains were obtained. A report has been prepared regarding the results of the activity. The Y. lipolytica wild-type strain does not produce glycolipid bio-surfactant substances. The ability of the Y. lipolytica wild-type strain to transform with a GoldenGate construct containing GFP was also demonstrated, and GFP was successfully produced. The level of sophorolipids in the recombinant strains did not reach the detection threshold. It is possible that part of the synthesis pathway is inhibited, or Y. lipolytica clones with suppressed sophorolipid synthesis have been selected. A report on the activity results has been prepared. In addition, the project team member participated in special international professional training on Y. lipolytica.
At the partner institution, JSC "Biotehniskais centrs", fermentation was optimized using the Model Predictive Control (MPC) system. The optimal composition of the fermentation feedstock was determined, and a prototype MPC algorithm was developed and tested. The obtained result will provide a foundation for further development and implementation of MPC systems in the production of bio-surfactant substances. The assessment of the algorithm's operation was conducted under conditions of input data disturbances (stability analysis of the algorithm). The main objective of the work package was achieved, and a report on the relevant results was provided.
During the reporting period, various dissemination and communication activities were carried out for the project. Results from the environmental impact assessment were published in a scientific article: Balina K., Soloha R, Suleiko A, Dubencovs K, Liepins J, & Dace E, 2023, “Prospective Life Cycle Assessment of Microbial Sophorolipid Fermentation”. This paper was published in the journal "Fermentation," [DOI:10.3390/fermentation9090839].
A presentation on the project results was delivered to industry professionals at the COST Action WIRE (Waste biorefinery technologies for accelerating sustainable energy processes - CA20127) scientific workshop held in Cottbus, Germany.
Overall, this project has made significant progress in the development of a scalable and sustainable biosurfactant production technology. The project team has made important
contributions to the scientific knowledge base on S. bombicola and Y.lipolytica metabolism and sophorolipid production. The developed genome-scale metabolic model and life cycle assessment study will be valuable resources for future research and development in the field, while the developed technology prototype is prepared for scale-up and application in biosurfactant production industry.
19.10.2023.