Interview with Jose M Sanz Martin, CARTIF

A central step to fully comply with the successful operation of the BioSFerA model is to ensure the highest yield of the fermentation steps by optimizing the different parameters that influence the biomass growth and its productivity.

In the project, CARTIF is one of the partners with an important role in the biological process for the lipids production at lab-scale. Thanks to its experience as Applied Research Centre Foundation in terms of R&D and technology transfer activities, CARTIF is leading the biological step referred to the project’s WP3 activity. Here, few questions to Jose M Sanz Martin, Researcher at CARTIF in the Agrifood and Sustainable Processes Division, to better understand the BioSFerA biological process development at lab-scale and the double-stages fermentation process that leads to the acetate before and after to the TAGs lipids production:

What are the last updates on the optimisation of the acetate fermentation process parameters for C14 and C16-18 TAGs production?

We are currently working on finding the best bioreactor strategy to favour lipid accumulation and for this we are testing nitrogen-limited conditions, continuous feeding of acetate as a main carbon source and recycling of cells using hollow fibre membranes. We are also using an engineered Yarrowia lipolytica strain developed by CSIC, which has improved triacylglycerides (TAGs) productivity. The results are promising and all this leads us to increase lipid production with respect to the wild-type strain.

 

Did you find any barriers and if yes, how are you trying to dealing with them?

Regarding the gas fermentation, one of the key aspects to improve the process is the choice of a suitable working strain, as well as the establishment of process conditions that allow for higher productivity. In addition, in order for the gases to be used efficiently by the microorganisms, it is very important that they are solubilised properly in the culture medium. To solve these problems, we have selected a strain of the acetogenic bacterium Moorella thermoacetica that is capable of producing a large amount of acetate from syngas, and we have improved the growth conditions by using bioreactors that allow us to control the flow rate of the gas fed as well as to increase the working pressure to favour its solubility in the medium.

As for acetate fermentation with Y. lipolytica, although acetate is a promising cost-effective and suitable carbon source for microbial fermentation, its use as the sole carbon source to enhance lipid synthesis is not very favourable from a metabolic point of view. To overcome this problem, we have confirmed that feeding a co-substrate such as glycerol can help to improve biomass production and drive metabolic flux towards TAGs production.

1.5 L continuous fermentations for TAGs production (installation of cell recycling system and continuous feeding strategy). Detail of the growth of Y. lipolytica from acetate+glycerol.

 

What are the last promising results?

In relation to acetate fermentation with Y. lipolytica, although we have not yet achieved the expected objectives of lipid production, we observed that the addition of glycerol during the fermentation process together with a nitrogen starvation strategy and limited stress conditions greatly favours the accumulation of TAGs by facilitating their metabolic biosynthesis. From here, we are conducting tests to find the most suitable concentration of glycerol to maximise this lipid accumulation.

 

For the overall biological production process of lipids from syngas at lab scale, several partners are involved in its optimisation and in different way. As leader of this activity, how this collaboration can impact the research and results?

I believe that the collaboration between all the partners involved in this work package is very beneficial for the progress of the project, as we can share the large amount of work that needs to be done according to the experience and capabilities of each partner. For example, VTT’s expertise in gasification has allowed us to select the best process conditions to achieve a syngas suitable for fermentation with microorganisms.  CSIC has obtained genetically modified strains of Moorella and Yarrowia that will maximise the productivity of the process, which is essential for the next stage of scaling up. In terms of bioprocess optimisation, BBEPP has focused more on optimising the anaerobic fermentation conditions, while CARTIF has focused on aerobic fermentation. Finally, ENVIPARK has contributed its expertise in the further processing of the lipids obtained in the fermentation broth.

In addition, the sharing of all the work done and the close collaboration that we have maintained throughout the project has allowed us to clarify many doubts and resolve the technical difficulties that we have encountered. It is certainly very gratifying to be able to work on this topic with some of the best researchers in Europe.

From 8th to 11th November, BioSFerA will take part at the ECOMONDO Exhibition, International fair dedicated to the circular economy and the ecological transition with base in Rimini (Italy).

An important annual event for circular economy experts from several sectors to meet each other and receive updates about the last technological innovations. BioSFerA will be there in different ways:

• virtual stand, from our partner’stand Environment Park, search for BioSFerA in its projects’ list;
• in-presence with some project’s representatives from Environment Park, scheduling B2B meetings from the virtual stand with each person;
• project presentation at the INDUSTRIAL&REPLICATION WORKSHOP, 9th November, 2-6 pm, organised by GLAMOUR project. The event aims to give last updates on the clean technologies for second generation biofuels, gathering experts and International research projects. Here the link to register at this hybrid event and the agenda.

 

For more information, please contact marianna.franchino@envipark.com

 

For the first time, after two years of project, the BioSFerA Consortium meets in person in Madrid. The first physical meeting took place last 3^rd – 4^th May at the Center for Biological Research Margarita Salas (CIB) of our partner CSIC, the Spanish National Research Council. The two-days meeting was also the opportunity to visit the CIB-CSIC’s laboratories, where a large number of researchers conduct their researchs on biotechnology, included the experiments for BioSFerA.

 

Concerning the CSIC’s activities, the research centre is continuing its trials currently dedicated to the genetic modification of selected acetogenic bacterial and oleaginous yeast strains: based on genetic engineering strategies, CSIC has created 8 recombinant Moorella strains with different acetate production rates. Results are still not fully satisfying, so that CSIC will move forward to better understand the recombinant bacterial behaviour, considering extreme fermentation conditions and syngas contaminants. On the other hand, genetic modifications on several genes involved in lipid metabolism are also in progress for Y. lipolytica, the oleaginous yeast used in the triacylglycerides (TAGs) production phase from acetate.

 

Meanwhile, the investigation of the gas & liquid fermentation processes for the parameters’ optimization are still ongoing. The activities were conducted on pressurized bioreactors of 1 L and 10 L dimensions for CARTIF and BBEPP respectively and using realistic data on the VTT’s syngas mix. They operated in different conditions to explore the factors influencing the acetate production, such as the use of different media or the application of a continuous fermentation mode with a membrane for cell recycling. A general finding from these tests is that increased biomass concentration can lead to higher volumetric acid acetic production. In addition, increased pressure was applied to overcome gas transfer limitations. Further investigations on the oleaginous yeasts’ fermentation step are also proceeding: BBEPP presented the results so far of acetate fermentation trials in the 7 L fed-batch bioreactor applying a pH-static feeding strategy, whereby different trials were performed using the both wild-type and genetically modified Yarrowia strains. CARTIF achieved tests using a fed-batch fermenter and two different continuous feeding strategies (high constant and gradually increased flow rate), but both the feeding and the cell recycling strategies need to be optimised to further increase TAGs production. First experiments were also performed by ENVIPARK with its steam explosion pilot plant as first step in the downstream process for the TAGs recovery. These first trials were conducted using a broth sample provided by BBEPP and literature parameters for an initial setting.

 

Even the preparations for the pilot process activities are advancing: VTT updates the Consortium with the successful accomplishment of the Factory Acceptance Test and the last steps to conclude the gasification unit, while BBEPP informed about the finalisation of the Bio Base Mobile Pilot Plant (BBMPP) construction. A draft timeline of the work has been presented, defining the transportation of the BBEPP’s mobile unit to the VTT site at the end of the year 2022 and the launch of the pilot process integration within 2023.

 

Within the progresses of the project’s activities, it is time for the scaling-up of the BioSFerA’s concept, starting with the DFB (Dual Fluidized Bed) gasification system as first block to help its functionality and transferability at industrial level. The techno-economic, environmental and social analysis has started as well. RINA, and the other partners involved in the activity, are preparing the basis for the future assessments related to the LCA/LCC analysis (CERTH), and the social-LCA too (NTUA). One reason of discussion was the current energy crisis and the impact of the COVID-19 outbreak on the expected techno-economic performance indicators, which will need to take into account these aspects.

 

Finally, the dissemination activities continue to follow the project’s progresses and advance with new activities, such as the release of the official BioSFerA’s newsletter at the end of May and the update of the website with the news related to the pilot process. On the 4^th May afternoon, after the meeting, a workshop on “Advanced Technologies for Green Molecules Production” was also organised in hybrid format in synergy with other two projects COS2MOS and Life Biomass C+, giving evidence of the first project’s results and its contribution to the biotechnology research development. After this fruitful meeting, the Consortium expects to meet again in October, this time in Belgium at the BBEPP’s headquarter.

The BioSFerA Consortium at CSIC premises during the 4^th GA meeting

Interview with José Luis García

Spanish Nation Research Council (CSIC), as one of the largest European public research institution with high expertise in the biotechnology field, gives its contribution to BioSFerA on biological processes at laboratory scale thorough its institute, the Center for Biological Research Margarita Salas (CIB) (Madrid). Its main activity is the research of the best microorganisms, playing a key role in the double stage fermentation characterising the BioSFerA process.

Exploring the micro level and the microorganisms’ interaction with the surrounding environment is crucial when moving processes from the laboratory to pilot scale level. Few questions to go deepen the CSIC activity and the biological research in laboratory with José Luis García, Research Professor at CIB (CSIC) and leader of the Environmental Biotechnology group:

 

As project partner, how CSIC contribute to the project and what is the innovative aspect led by your research activity?

Our main contribution to BioSFerA is to develop a new collection of system metabolic engineering tools that can allow to modify the acetogenic bacteria and the oleaginous yeasts in order to obtain robust bacteria and yeast that could render higher production yields of acetate and lipids than the natural wild type strains. Using these tools and considering the lessons learned during the implementation of the processes at industrial scale it will be possible to propose other modifications to improve even further the production yields.

 

Your work is focused on bacteria and their interaction with organic matter and the surrounding environment. What are the main limitations hindering bacterial activities to consider for their performance, specifically in the BioSFerA processes?

It’s important to consider the final objective of the process, which in BioSFerA is to transform the syngas obtained from biomass gasification into acetate that will be further used to produce lipids. Analysing our model, we can split it into two main steps: in the first one, we use acetogenic bacteria to transform syngas into acetate, whereas in the second step we use oleaginous yeasts to transform acetate into lipids. To develop an industrial efficient process the first problem that we have to handle is how to increase the bacterial production of acetate from syngas. This can be achieved by combining the production of enough microbial biomass from syngas and an efficient transformation of syngas into acetate. In addition, we have to take care of possible syngas contaminants that are toxic for the bacterial acetogenic strains and that can affect bacterial growth or viability. Hence, we have performed a screening of different acetogenic bacteria that are able to grow at high cell densities, that produce high amounts of acetate, and that are resistant or tolerant to some contaminant/impurities present in syngas.

Example of Y. lipolytica strains growing
on YPD (rich liquid medium) agar plates.

 

Considering the acetate production, how the syngas composition from the different biomass can affect the fermentation process?

As mentioned above, in BiosFerA we are traying to transform the residual biomass into syngas: a mixture of carbon dioxide, carbon monoxide and hydrogen, that is obtained by a process named gasification. The syngas, as result of a gasification process, depends on the origin and properties of the biomass which influence the presence of the contaminants and the syngas composition. Usually, the biomass gasification not only produces syngas, but also other compounds like hydrogen sulphide, hydrogen cyanide, benzene, ammonia, methane, etc. This means the production of acetate will be affected by the proportion of the main gases (CO2, CO, H2), that varies depending of the biomass and the gasification conditions and that are responsible of the bacterial growth and the acetate yields, and also by the presence of toxic unwanted contaminants. Therefore, it is important to find the most appropriated syngas composition as well as to eliminate as much as possible the unwanted contaminants to a level that can not affect bacterial growth or acetate production. 

 

What kind of approach have you followed in these research activities and how is the work coordinated with the other partners? What are the advantages of having such a broad, interdisciplinary team as BioSFerA?

In a very early steps, we have collected all the information available in the literature dealing with the metabolism, molecular biology and processes of the organisms of interest, this is acetogenic bacteria and oleaginous yeasts. Then, we have selected the best strains and performed a screening of their properties using different culture conditions in order to test which are the best acetate producers from syngas and lipid producer from acetate. After selecting the best performing microorganisms, i.e. the acetogenic bacteria Moorella thermoacetica and the oleaginous yeast Yarrowia lipolytica, we have used systems metabolic engineering approaches to identify the target genes that have to be deleted or overexpressed, to channelling the metabolism to the production of the metabolite of interest. After modifying the strains, they have been initially tested at CIB-CSIC at lab scale using shake flask cultures and then transferred to CARTIF and BBEPP for fermentations in bioreactors at high volume levels. At this point, the interdisciplinary nature of the different groups involved in BioSFerA project allows us to find the best operational conditions to produce the compounds of interest at pilot and industrial scales.

 

Acetate metabolism in acetogenic bacteria.
Target genes are indicated in colours.

Lipid (TAG) metabolism in oleaginous yeasts.
The main target genes are indicated in red.

 

So, first you analyse the organism’s metabolism to identify the best strains and then you guide them in the metabolic process of the desired substances, through genetic modifications. What is the value added to use GMO microorganisms?

GMO microorganisms are designed for different purposes, but in principle they are all aimed at increasing the performance of processes. The yield of the process can be increased by redirecting the carbon flux to the production of the desired product and at the same time by avoiding the formation of unwanted by-products. Additionally, as it is the case of lipids, we can also modify the microorganism eliminating the degradative pathways of lipids and fatty acids, facilitating that the decided final product is accumulated inside the cell, without the possibility of its further degradation. The GMOs constructed in BiosFerA project are oriented to increase the production of acetate from syngas in the case of acetogenic bacteria, and to increase the production of lipids from acetate in the case of oleaginous yeast. If required, the strains can be also modified to acquire other properties, for instance the resistance or tolerance to specific toxic contaminants.

Electroporator used to create OGMs

 

Detailed information on the biological research at laboratory scale carried out by the BioSFerA’s partners are available in the Deliverable 3.2 – Deliverable 3.3.

Further explore through the opinion-Editorial Letter by CSIC – Integrating greenhouse gas capture and C1 biotechnology: a key challenge for circular economy.