Project concept

It is well known that the reserves of oil, natural gas and coal are limited. In contrast, biomass is a reliable resource for fuels and chemicals in the long term[1]. Supplementing petroleum consumption with renewable biomass resources is of critical importance in sustaining the growth of the chemical industry. The advantages of using biomass rather than petroleum to manufacture chemicals include opportunities for less pollution, no net CO₂ contribution to the atmosphere, more biodegradable and sustainable products and, in some cases, lower cost[2]. It has been found many biomass derived chemicals have economical advantages, particularly for some functionalized chemicals [3]. In addition, recent advances in fermentation technologies such as enzymatic engineering, metabolic engineering and genetic manipulation, provide new opportunities for producing a wide variety of industrial products from renewable plant resources[4,5]. A key to the chemical industries gradual shift toward the use of renewable biomass resources is the implementation of the bio-refinery concept[6]. Similar to a petroleum refinery, a bio-refinery integrates a variety of processing technologies to produce multiple bio-products from various bio-masses. Two of the 12 molecules that have been identified as most promising chemical building blocks that can be obtained from biomass containing sugars are levulinic and succinic acids.

Romania is a country with a well developed forestry sector and high surface of agricultural land both generating a wide range of biomass resources with an exploitable content of sugars. The assessment of raw material cost and the estimation of the potential market size clearly shows that the current petroleum based succinic acid process will be replaced by the fermentative succinic acid production system in a near future. The same is valid for levulinic acid for which the chemical conversion of cellulose or starch can be done with high efficiency and low costs.

This project's aim is to develop a set of technologies for converting available biomass generated by wood industry and/or agriculture into levulinic and succinic acids. The novelty elements of our approach include:

• synthesis of levulinic acid we will start from biomass rather than pre-processed sugars as most of the of the literature data suggests;
• process designed to accept different types of biomass in the same equipment by using ultrasound to enhance mixing and cell wall breaking. Wood saw or shavings, potatoes and corn where taken into account. It is worth mentioning that in the last years in Romania corn was cheaper than wood and the over production of potatoes was left in the ground;
• levulinic acid conversion to succinic acid based on heterogeneous catalysis as a backup route to the preparation of succinic acid by fermentative processes;
• design of a solid acidic catalyst for the sugars to levulinic acid conversion to, avoiding in this way expensive neutralization stages and acidic waste waters;
• isolation of GM E. coli strains able to convert sugars and/or glycerol to succinic acid.

Based on these statements the main objective of this project is to develop a set of technologies for the Synthesis of some C4, C5 carboxylic acid building block chemicals from renewable biomass resources. Starting from here the project will aim for the following derived objectives:

1. Development of a process for levulinic acid production from three different renewable bio-resources: wood, potatoes and corn.
   This will be done by CO and P1 and include:
   • design and synthesis on new solid catalysts and conversion process. Magnetically recoverable nano-catalysts will be considered as main option;
   • ultrasound assisted process enhancement through mixing and cell wall breaking;
   • process parameter optimization for wood biomass to preserve lignin. Such a step is essential in the design of a complex bio-refinery in which both cellulose and lignin should be valorised. This is also an element of novelty of our approach.
2. Novel chemical procedures for conversion of levulinic acid and reaction by-products to succinic acid and methyltetrahydrofurane (MTHF).
   This will be done by P1 and CO and will include:
   • catalytic conversion of furfural to succinic acid. Furfural is a main by product of levulic acid production through acid hydrolysis of polysugars;
   • catalytic conversion of levulinic acid (or HMF) to succinic acid;
   • catalytic hydrogenation of levulinic acid (LA) to methyltetrahydrofurane (MTHF).
   Catalysts prepared via the hydrolysis of ruthenium chloride to Ru(III) deposited on aminofunctionalized silica-coated magnetic nano-particles and reduced Ru(0) active species deposited on the same type of magnetic particles will be used as green alternative.
3. Glycerol or glucose conversion to succinic acid by fermentation. This will be carried out by P3 with analytical support from CO and P1.
   This objective will include:
   • Isolation of 2 different genetically modified E. coli strains for succinic acid production designed for consuming glycerol and/or glucose. Initial selection will be made from Sapientia University's collection of microbial cultures which includes about 300 strains. The best producing E. coli strains will be genetically engineered by deletion of two genes, with the method of Datsenko [60], which are responsible for the catalysis of succinate production competing metabolic pathways for. The first deleted gene will be plfB (pyruvate format lyase) and the second the pst1, a gene from the phosphotransferase system of the bacteria. The pyruvate formate lyase deletion is expected to decrease the byproducts production, esspecially ethanol and acetate. The second gene deletion will inactivate the phosphotransferase system, therefore more phosphoenol pyruvate (PEP) flows to succinate than to pyruvate.
     A similar procedure will be used to obtain a glucose consuming GM E. coli: the plf (pyruvate formate lyase) gene, the lactate dehydrogenase (ldh) and/or the alcohol dehyprogenase (adh) genes will be deleted;
   • Optimization of culture condition in bioreactor for GM E. coli strains;
   • Determination of the best environmental and genetic conditions for succinic acid production in Mannheimia succiniproducens. Known as one of the best succinic acid producers, this bacteria will be studied in base of an in silico metabolic flux analysis, the results will be verified experimentally, and finally the best conditions for succinic acid production in bioreactor will be determined. This will also include: total genome analysis and in silico metabolic flux analysis , transcriptome analysis using DNA microarray techniques to measure the expression levels of genes implied in different metabolic pathways of succinic acid production, by detecting cDNA after reverse transcription of mRNA of the genes;
   • Selection of the most efficient strain and optimization of the culture conditions in bioreactor.
4. Design, set-up and operation of a mobile pilot plant unit for the conversion of biomass to crude levulinic acid. This will be carried out by the Industrial partner P3 in cooperation with CO. In our view this unit will be able to work close to the biomass source and convert it to a levulinic acid containing fluid mass that can be stored and transported in tanks to a single workup facility. This is needed because biomass will be generated by relatively small companies in quite spread locations;
5. Economic evaluation of the efficiency of levulinic acid production from three types of biomass : wood, potatoes and corn. This will be done by P4;
6. Dissemination of results at local and international level.