PhD-project: Development of an in vitro method for measuring digestibility of protein-rich raw materials in Atlantic salmon

Martin Larsen, Faculty of Chemistry, Biotechnology and Food Science, NMBU.

Period: March 2023 – February 2027

Supervisor: Sabina Leanti La Rosa (NMBU).
Co-supervisor: Vincent Eijsink (NMBU), Svein Horn (NMBU), Piotr Lisik (Skretting) and Gunvor Baardsen (Skretting).

Project description
The overall objective of this PhD project is to develop an in vitro method to assess digestion of protein-rich raw materials for salmon feeds. This system will simulate conditions of the different sections along the salmon gastrointestinal tract and will eventually be used as a pre-screening tool to determine the digestibility of proteins in new feed ingredients.

The initial focus within the project will be proteomic analysis of the contents of the stomach, pyloric caeca and midgut in salmon fed protein-rich diets. This analysis, supplemented with enzyme activity measurements, will reveal prevalent active proteases released in the three compartments, including potential microbial proteases.

A large portion of the work will focus on the extraction of digestive enzymes from salmon intestines, with development of suitable procedures for purification, as well as short- and long-term storage of the enzymes and enzyme cocktails. Further, key proteases identified will be cloned, produced and characterized in detail. Also, the in vitro digestion method will be developed optimized with respect to enzymes, enzyme mixtures and robust protocols for feed- break-down simulation.

The project is central in SFI-IB project P4 and is a collaboration effort with SFI’s industrial partner Skretting.

PhD-project: Automated strain design using genome-scale models and multi-omics data integration

Elisa Márquez-Zavala, PhD student, NTNU, Department of Biotechnology and Food Science.

Period: January 2022 – December 2024

Supervisor: Daniel Machado, NTNU, Department of Biotechnology and Food Science.
Co-supervisor: Francesca Di Bartolomeo, SINTEF, Department of Biotechnology and nanomedicine.

Project description
Genome-scale metabolic models (GEMs) represent the complete metabolism of an organism, accounting for thousands of metabolic reactions that are encoded in its genome. These models allow us to simulate the effect of genetic and environmental perturbations in the production of target chemical compounds. This is a toolbox we can use to optimize microbes for industrial applications.

In this PhD project, the goal is to make GEMs as a standard framework for computational design of microbial cell factories. The objective of this is to develop common protocols and workflows that meet the different needs of industrial partners as well as have a wide range of applications. As GEMs still pose limitations, e.g. that they do not account for other biological processes and the different layers of cellular regulation, focus will also include newer modeling approaches and more advanced simulation methods that can overcome these limitations. In the age of Big Data, ways to integrate various types of so-called omics data with metabolic models will also be explored.

This project is primarily part of SFI-IB Project 1.

PhD-project: Development and application of fluxomic tools to develop microbial cell factories for industrial cultivation conditions

Divyata Vilas Rane, PhD student, NTNU, Department of Biotechnology and Food Science.

Period: January 2022 – January 2025

Supervisor: Professor Per Bruheim, NTNU, Department of Biotechnology and Food Science.

Project description
This PhD project aims to understand the behavior of several microbial model systems under industrial processing conditions using MS-based metabolite and fluxome profiling. The model systems include Escherichia coli for recombinant protein production and actinomycetes strains for antibiotic production.

Established mass spectrometry-based quantitative metabolite profiling and 13C/15N-label incorporation methods will be central in the project and collected data will be used to optimize industrial fermentation processes and to obtain comparative data between lab-scale and industrial processing conditions.

In the initial phase of the project, recombinant protein production in E. coli has been in focus, in particular a study on adaptation at central metabolism level during transition from oxygen surplus to oxygen limiting conditions. Oxygen limitation is challenging in industrial level cultivations, and by increasing knowledge of how the cells respond and adapt to such conditions, strains and cultivation conditions able to maintain high productivity might be in scope of development.

This PhD project is associated SFI-IB Projects 1 and 3 and in collaboration with SFI industrial partners Vectron Biosolutions and Xellia Pharmaceuticals. However. cultivation and analytical technology established will be generic and can be applied on several industrial microbial bioprocesses.

PhD-project: Optimized bulk starter cultures for cheese production

Inga Horntvedt Thorsen, PhD student, NTNU, Department of Biotechnology and Food Science.

Period: April 2022 – April 2026

Main supervisor: Professor Per Bruheim, NTNU, Department of Biotechnology and Food science.
Co-supervisors: Jorun Øyaas, TINE SA, and Håvard Sletta, SINTEF, Department of Biotechnology and nanomedicine.

Project description
This PhD project aims to understand causes for variation in the production of bulk starter cultures for cheese production. This include studies of how starter cultures are affected by e.g. growth media during production of the starter culture. Monitoring of how the number and distribution of bacteria in the mixed consortia vary at species and sub-species levels with variations in the bulk start conditions is of particular interest. Samples form dairy plants will be included to examine variations at the industrial site using laboratory scale studies.

The information and knowledge from these experiments will be used to improve process control and cheese quality and contribute to reduce waste and production costs. Initially, this project has focused on evaluation of the starter cultures, which are developed for industrial dairy usage and are bought directly from the commercial producers, and these typically contain hundreds of strains but with a limited number of dominant species. This preliminary testing has been important to establish robust protocols for handling of these mixed consortia, adjusted not to introduce biases and keep conditions relevant for transfer of knowledge to the industrial scale.

The project is primarily linked to SFI-IB Project 1 and partner TINE AS and is also financed by TINE AS and the NFR NæringsPhD program.

PhD-project: Study and exploitation of the T3 secretion system in Escherichia coli

Antonia Areali, PhD-student, NTNU, Department of Biotechnology and Food Science.

Period: October 2021 – October 2024

Supervisors: Trygve Brautaset and Fernando Pérez-García, NTNU, Department of Biotechnology and Food science

Project description
In this PhD project, the T3 secretion system (T3SS) will be explored. T3SS is a one–step secretion system already applied in the production of proteins with industrial relevance using common microbial platforms such as Salmonella and Escherichia coli. Protein secretion is essential for prokaryotes and is also highly relevant for industrial bioprocesses as it transports a produced cellular protein across the membrane. T3SS is known to secrete protein very fast and efficiently, however, the system is seemingly limited regarding the size of the proteins secreted, and hence it is key to investigate this system in more detail to optimize an industrially competent exploitation of T3SS.

In the project, reporter proteins will be applied to study secretion as these are easy to measure/ monitor. Examples are reporters as fluorescent proteins (e.g., green fluorescent protein(GFP)) or enzymes (e.g., amylases). By rationally modifying the size of those proteins, we intend to shed light on the molecular limitations of the T3SS as well as identify possible targets for further improvements of the secretion system.

This project is primarily linked to SFI-IB project 3, and is also relevant for SFI-IB Industrial partners Vectron Biosolutions and Arcticzymes.

PhD-project: Techno-economic evaluation of biochemical processes – systematic methods for optimal design

Minglin Xu, PhD Student, NTNU, Department of Chemical Engineering.

Period: September 2021 – 2024

Supervisor: Magne Hillestad, NTNU, Department of Chemical Engineering.

Project description
The overall objective of this PhD project is to develop a techno-economic assessment tool that allows convenient and accurate assessment of novel biochemical processes. This tool will consist of four parts, i) process models, ii) simulation and optimization tools, iii) commercial cost data, and iv) economic models. The project will include techno-economic assessment of specific cases from the industrial partners within SFI-IB, and the developed tools will be used to assist the analysis of the new biochemical technologies.

The first focus within the project has been on establishment of a mathematical model for an anaerobic digestion process and simulation of a biofilm plug flow reactor application in biomethane production (as a case for one SFI-IB partner). Further, the anaerobic digestion process is optimized by conducting a systemic approach to determine the optimal reactor configuration and reaction conditions.

This project mainly belongs to SFI-IB Project 6, however it will be relevantfor several of the other Projects, depending on the cases applied.

PhD-project: Interface mass transfer and bubble hydrodynamics in complex fluids

Hauna F. Hosen, PhD Student, NTNU, Department of Chemical Engineering.

Period: September 2021 – September 2025

Supervisor: Jannike Solsvik, NTNU, Department of Chemical Engineering.
Co-supervisor: Hugo A. Jakobsen, NTNU, Department of Chemical Engineering.

Project description
The aim of this PhD project is to improve our understanding of flow dynamics and transport phenomena in the dispersed multiphase bubble-in-liquid fermenters. For optimal design, scale-up, and operation of gas fermenters, better knowledge is needed on bubble hydrodynamics in various flows and fluid properties, as well as how the various conditions influence the bubble-liquid interfacial mass transfer rate. These described phenomena will be investigated in bubble columns and stirred tanks and Non-Newtonian rheology and nutrients typically found in broths will be analyzed.

The project is mainly experimental and high-speed imaging is one of the central techniques applied. Collected experimental data will be analyzed by means of e.g., machine learning techniques (deep neural networks), and the data will be compared with mathematical simulations when appropriate. 

This project is part of SFI-IB Project 5 on gas fermentation and also involves established collaboration with other (international) research groups.

PhD-project: Design, production and testing of novel prebiotics based on lignocellulosic materials

Lars J. Lindstad, PhD Student, NMBU, Faculty of Chemistry, Biotechnology and Food Science.

Period: 2019 – 2024 (incl parental leave)

Supervisors: Bjørge Westereng NMBU, Faculty of Chemistry, Biotechnology and Food Science.
Co-supervisors: Sabina Leanti La Rosa, Shaun Leivers, Phillip Pope and Siv Kjølsrud Bøhn.

Project description
This PhD project focuses on exploring how complex carbohydrates are utilized by gut bacteria as well as production and testing of new potential prebiotics, with a focus on the human gut and bacteria that are important for human health and well-being. Substrates used are hemicelluloses from spruce and birch that resemble, but are often more complex than, mannans and xylans found in food, and thus may be used to target some of our commensal bacteria.

The project will include enzymatic production, modification, and precise characterization of hemicellulose-derived oligosaccharides and testing of the impact of such oligosaccharides on single bacterial cultures and bacterial communities in small fermenters. Discovery and characterization of new enzymes for tailoring the prebiotics is another important element in the project.

The work aims to understand the enzymatic machineries that bacteria use to process complex carbohydrates, work that may result in novel enzymes, potentially also be useful for wood biomass processing in other contexts. Finally, both enzymatic and chemical methods will be applied to modify carbohydrates, not only for changing functionality and complexity, but also to understand and track, in vivo, how they are utilized.

This project is related to SFI-IB projects P3 & P4 and the most important industrial partner is Borregaard.

PhD-project: Development of enzymatic tools for processing of chitin-rich biomass

Amanda Votvik, PhD student, NMBU, Faculty of Chemistry, Biotechnology and Food Science.

Period: 2020 – 2024

Supervisors: Morten Sørlie, NMBU, Faculty of Chemistry, Biotechnology and Food Science
Co-supervisors: Vincent Eijsink, Zarah Forsberg

Project description
This PhD project involves enzyme discovery and engineering, with the aim of developing advanced enzyme technologies for cost-effective biorefining of chitin-rich biomass to produce valuable products like defined chitooligosaccharides (COS) and N-acetyl glucosamine (GlcNAc). Novel enzymatic technologies will be explored, e.g. the catalytic enhancement of minimal chitinolytic enzyme cocktails through addition of lytic polysaccharide monooxygenases (LPMOs) and the controlled supply of H₂O₂ under anaerobic conditions.

Thus, the project focuses both on finding optimal (combinations of) enzymes as well as optimizing bioprocessing conditions, resulting in high efficiency and desired product profiles. The enzyme engineering work focuses on the interplay between the processivity of chitinases, which will be manipulated, and enzyme efficiency during bioprocessing.

The project belongs to SFI-IB projects P3 and P4 and is relevant for multiple SFI-IB industrial partners such Nutrimar and Biocatalysts. The project is also connected to a new EU project staring up in September 2022 called Valuable (VALorisarion of fUngAl Biomass using noveL Enzymatic technology).

PhD-project: Biotransformation of feathers rest materials

Clémentine Isembart, PhD student, NMBU, Faculty of Science and Technology

Period: 2022-2026 (started in October 2022)

Supervisors: Volha Shapaval, NMBU, Faculty of Science and Technology
Co-supervisors: Achim Kohler, Boris Zimmerman, Vincent Eijsink, Svein Horn, Marije Oostindjer (Norilia)

Project description
This project focuses on development of microbial and/or enzymatic conversion technologies to convert feathers into sustainable high-value bioproducts. The PhD project aims at providing a detailed understanding of which filamentous fungi and bacteria that are able to degrade keratinous biomass, and why. At one hand, optimization of microbial conversion will be targeted, e.g., optimal process parameters for microbial and enzymatic conversion of feathers, and on the other hand, key enzymes will be identified and analyzed concerning their possible development into industrial biocatalysts. When it comes to microbial processes, monitoring of optimized fermentation/digestion processes by online monitoring and vibrational spectroscopy will also be assessed.

This project belongs to SFI-IB projects P3 and P4, with focus on P4. One co-supervisors is from SFI-IB Partner Norilia and other (potentially) involved SFI-IB companies include Nutrimar and Biocatalysts.

Postdoc-project: Development of a platform for efficient fungal expression of enzymes

Yogesh Chaudhari, Post-doc, NMBU, Faculty of Chemistry, Biotechnology and Food Science

Period: 2021 – 2024

Supervisors: Vincent Eijsink, NMBU, Faculty of Chemistry, Biotechnology and Food Science
Co-supervisor: Svein Horn, NMBU, Faculty of Chemistry, Biotechnology and Food Science

Project description
This project focuses on production (up to gram amounts) of enzymes selected by the industry partners and/or enzymes needed in SFI-IB Project 4 (novel bioprocessing scenarios) in Trichoderma and or Aspergillus production systems. In addition, it will contribute to efforts for production of fungal single cell protein. Initial work has involved cloning and enzyme production, yielding active enzymes produced at some 2 g/L during lab scale (flask) cultivations.

However, yields are likely to be highly improved when applying cultivation in fermenters. Further, through a highly interactive process with SFI-IB industry partners, a list of target enzymes has been established and the expression and purification of these enzymes is a high focus in the project.

The project is central in SFI-IB project P3 and there are interactions with multiple industrial partners, including Borregaard, Norilia, Biocatalysts and Arcticzymes.