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Valorisation of biorefinery by-products leading to closed loop systems with improved economic and environmental performance

Final Report Summary - VALOR-PLUS (Valorisation of biorefinery by-products leading to closed loop systems with improved economic and environmental performance)

Executive Summary:
The project Valor-Plus targets the generation of bulk and high value product streams from the byproducts lignin, hemicellulose and glycerol by developing new science and technologies, and utilising a platform of integrated technologies. The platform technologies will enable the optimum value to be achieved for each byproduct stream whilst ensuring cost-effective and complete utilisation of available resources.

The project will therefore enable the realization of sustainable, economically viable closed-loop integrated biorefineries. Valor Plus will lead to the design, development and integration of new processes to form closed-loop systems to make the best use of biorefinery by-products and deliver new high value products with improved economic and environmental performance. To this end, it will provide new knowledge, new biotechnologies and new products that support the release, refinement and transformation of lignocellulosic biomass lipids and low volume functional chemicals to produce multiple bulk and high value product streams, allowing for the full use of the biomass with zero waste and thereby generating the highest value return.

The project is divided into five main development streams. The pre-treatment and fractionation focuses on the development of a novel methodology for the controlled breakdown and the release and fractionation of the lignocellulose biomass. The hemicellulose valorisation focuses on the engineering of new enzymes and microorganisms for the controlled hydrolysis and transformation of hemicellulose to high value oligomers and bulk fermentation product streams. Lignin valorisation targets the utilisation of combined chemo-enzymatic and chemo-microbial processes for the controlled depolymerisation and transformation of standardised lignin feedstocks to value product streams. The valorisation of glycerol is studied by engineering of new microorganisms suitable for the fermentation of crude glycerol to higher value product streams. Finally the technological and economic potential is demonstrated by the devolopement of demonstrating component technologies, roadmaps for technology and product stream integration, case studies and a full life cycle assessment.

Project Context and Objectives:
The Valor-Plus supports the realisation of sustainable, economically viable closed loop integrated biorefineries through the development of new knowledge, (bio-) technologies and products that enable valorisation of key biorefinery by-products.

Biorefineries are facilities that convert biomass feedstocks into bio-based energy, fuels, materials and chemicals using green / sustainable processes. Biorefineries offer a long term potential to meet our global resource needs while combating the effects of climate change.

The processes for the production of biochemicals, biomaterials and bioenergy generate vast streams of by-products such as hemicelluloses, lignin and glycerol which are currently widely underexploited due to the difficulties of their extraction and posterior transformation. The sustainability and economic viability of the biorefinery concept call for the exploitation of by-products streams and the development of closed loop systems.

The objective of this project is to develop physicochemical technologies which are concomitant to the enzymatic/microbial processes for product separation and purification as well as biotechnology approaches for the conversion of biorefinery by-products into added value bio-based products. These would include chemicals and chemical building blocks, biopolymers, materials, bioactive compounds and others. The European added value lies in increasing the effectiveness and efficiency in the value chain of bio-based products by reducing waste and generating higher value products or services.

With funding from the EU FP7 programme, the Valor-Plus project consists of a strong consortium of 14 partners, including SMEs, research centres, universities and one large enterprise. In delivering improved sustainability, commercial and environmental benefits, the Valor-Plus project is focused on five key areas:

1) Pre-treatment and fractionation: development of a novel methodology for the controlled breakdown, release and fractionation of the lignocellulose biomass.

2) Hemicellulose Valorisation: engineering of new enzymes and microorganisms for the controlled hydrolysis and transformation of hemicellulose to high value oligomers and bulk fermentation product streams.

3) Lignin Valorisation: utilisation of combined chemo-enzymatic and chemo-microbial processes for the controlled depolymerisation and transformation of standardised lignin feedstocks to value product streams.

4) Glycerol Valorisation: engineering of new microorganisms suitable for the fermentation of crude glycerol to higher value product streams.

5) Demonstration of the technological and economic potential: demonstrating component technologies, roadmaps for technology and product stream integration, case studies and a full life cycle assessment.

Project Results:
WP1: By-Products Quality Control
The main objectives of WP1 were the straw processing for by products and quality control of by products.

In summary the main achievements are:
The use of large conventional co-rotating intermeshing twin screw extruder with wider inlet demonstrated higher production efficiency for the low bulk density cellulosic feed stocks, like wheat straw.
The improved pre-treatment and post-conditioning during cellulosic biomass extrusion enhanced the quality and extraction rates of the hemicellulose, and established the base for our lab scale extraction system able to supplier hemicellulose in kgs.

The large scale isolated hemicellulose obtained from the upgraded twin screw extrusion processing technology were supplied to TUM and other our project partners and their analysis demonstrated higher standard and stable quality of hemicellulose obtained by Brunel University London.

A larger Werner Pfleiderer twin-screw extruder was refurbished and successfully operated for processing lignocellulosic biomass. The wheat straw was extrusion processed at a rate of 5kg/h which is much higher than we achieved when using Betol TS 40 twin screw extruder. The Werner Pfleiderer twin-screw extruder has larger screw diameter and also comes with a wider opening at feeder port which allows the possibility to process much more lignocellulosic biomass at any given time and it also makes it easy to deed the biomass continuously at the feed port.

A new pre-treatment procedure which was first tested during Technology of University of Munichs (TUM) PhD student’s working visit to UBRUN in 2016 was also adopted. A higher level of alkali was used to soak the wheat straw before it was extrusion processed and the extruded straw was conditioned in an oven for further treatment.

We were able to maximise our processing conditions and were able to process at rates not possible before. A minimum of 200g of dry hemicellulose was extracted from 1 kg of wheat straw. Preliminary enzymatic hydrolysis test were carried out by TUM; the results demonstrate that the produced hemicellulose at larger scale, i.e. processing in kgs. rather than milligrams and few grams scale with very good specific activity.

We concentrated our work on extrusion pre-treatment of wheat straw since it was readily available, and we are confident that similar results would be obtained if pine and spruce flakes or other feed stocks were used.

Quality control tests and measurements have been conducted by using tests e.g. FTIR, TGA and DSC to determine the consistency and quality of hemicellulose products. The improved process involved extrusion fractionation at 100 and 120 °C and post-extrusion conditioning treatments of the cellulosic biomass. Through steps of isolation, extraction and purification, hemicellulose with quality and large quantity (kgs supply) was successfully established during the period in our lab experiments.

WP2: Hemicellulose hydrolysis
Work package 2 focused on the degradation of hemicellulose from biorefinery by-products such as wheat straw, corn stover, and forestry wood to value-added products. These products encompassed monosaccharides and organic acids which can be used as a feedstock for fermentation processes to produce biofuels, and oligosaccharides, which can be added as valuable and health-promoting ingredients to functional foods. The structure of hemicellulose can be very diverse, being comprised of a wide range of different sugars and organic acid substituents, which must be considered when developing a technical process for hemicellulose degradation. The research endeavour within WP2 therefore included the development of an enzyme toolbox for the complete and selective enzymatic hydrolysis of hemicellulose, a filtration process for the purification of hydrolysis products and a chromatographic technique for the separation of hemicellulose hydrolysate constituents. Furthermore, WP2 included the evaluation of the suitability of provided hemicellulose preparations for enzymatic degradation to monosaccharides as feedstock for fermentation processes, as well as an evaluation of the potential of hemicellulose-derived oligosaccharides as high value products for application, e.g. as prebiotics in functional foods.

In order to find industrially viable enzymes specifically suited for the degradation of hemicellulose, five strains of well-known thermophilic, hemicellulose-degrading microorganisms were selected. Genes with proposed involvement in hemicellulose hydrolysis of these organisms were cloned and heterologously expressed, and the enzymes obtained were screened for their catalytic activities. More than 100 different enzymes were tested on polysaccharides, oligosaccharides and artificial substrates, finally constituting a well-defined hemicellulase toolbox.

The focus of the enzyme toolbox was set on two substrates: arabinoxylan and glucomannan. Both substrates represent abundant polysaccharides among hemicelluloses and therefore represent suitable model substrates to assess the enzyme toolbox’s potential to degrade hemicellulose. Based on this enzyme toolbox, suitable enzymes and optimized enzyme mixtures for an industrial application were selected and tested both at small and large scale. The characterisation covered classical biochemical but also process relevant parameters such as feasibility of efficient enzyme production, process stability, synergistic effects between enzymes, substrate recognition, and product specificity. The specific activities for all substrates, optimal enzyme concentrations as well as the pH and the temperature optima of the most promising enzymes for the degradation of arabinoxylan were determined.

The thermal inactivation over time of four enzymes which had been selected as candidates for the complete hydrolysis of hemicellulose were determined at 60 °C over a period of 48 h, after which >60 % of the initial activity was retained, thus displaying the anticipated enzyme stability. The substrate and product specificities of 38 enzymes were determined by analysis of their degradation of xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS). Differences in the degradability of AXOS by different xylanases were identified, which has to do with the fine structure of AXOS. Only few Arabinofuranosidases were able to degrade AXOS containing twofold arabinosylated xylose, which indicates that these oligosaccharides represent potential hydrolysis bottlenecks when aiming at complete hemicellulose hydrolysis. For the production of oligosaccharides from arabinoxylan and glucomannan, 84 enzymes from five different organisms were screened. Three different product patterns from xylanase action on arabinoxylan and five different product patterns from glucomannan degradation were identified that can be used to produce different XOS, AXOS and GMOS product mixtures.

Using the enzymes characterized in WP2, hemicellulose hydrolysis reactions were performed in sub-milliliter scale at first. They were scaled up to the litre-scale in a prototype hydrolysis at the end of the project. Different technologies such as chromatography, crossflow filtration, precipitation and successive solubilisation steps using ethanol-water mixtures were applied and adjusted to overcome the unique purification hurdles encountered during oligosaccharide purification. After optimization of each method, a downstream processing workflow for oligosaccharide purification was set up that meets the high requirements regarding product purity and product yield in industrial scale. A crossflow filtration system was designed and adjusted for the purification of oligosaccharides and performed in litre-scale with arabinoxylan and glucomannan hydrolysates. The method allowed the removal of particles, non-degraded polysaccharide and proteins, as well as the concentration of desired oligosaccharides. In order to develop a chromatographic method for the separation of oligosaccharides, different stationary and mobile phases and different chromatographic system configurations such as continuous and semi-continuous chromatography were tested and optimized. The optimized methods allowed the fractionation of oligosaccharides with narrow size distributions for manno-, glucomanno-, xylo-, and arabinoxylo-oligosaccharides and were shown to be suited to remove further contaminants such as polysaccharides, monosaccharides, and proteins. Similarly, the semi-continuous chromatography method developed in WP2 could also be used to directly purify xylitol from cell-free fermentation broth. Xylitol is a high-value product, i.e. a sugar substitute, formed from hemicellulose-derived xylose by fermentation, which represents a further pathway for hemicellulose valorisation that was developed in the framework of Valor-Plus.

Arabinoxylooligosaccharide and glucomannooligosaccharide samples that were produced from the hemicellulose substrates arabinoxylan and glucomannan, respectively, were purified in the described workflow and tested for prebiotic properties using different “beneficial” and “non-beneficial” test strains.

Finally, based on the multitude of recombinant enzymes characterized in WP2, several enzymes suited for hemicellulose hydrolysis were selected for use at a large scale. A fed-batch enzyme production process in a bioreactor and a purification procedure for these enzymes was established and implemented to supply these enzymes for arabinoxylan degradation. 40 different hemicellulose preparations were evaluated and the best preparation was selected. A prototype hydrolysis of hemicellulose with a combination of five enzymes was optimized by improving the enzyme concentration and reaction conditions such as pH and temperature. The targeted degradation yield of over 80 % was achieved by an enzymatic hydrolysis of a solution with a 1 % hemicellulose preparation. A prototype hydrolysis was carried out in litre scale using a bioreactor. Hydrolysates with the liberated monosaccharides were provided to WP4 for prototype fermentation.

WP3: C5 Sugar Fermentation
The different groups addressed different types of microorganism to address the common objective. In Brunel the focus was Gram negative bacteria, in TUM it was soventogenic Gram positive Clostridia, and at Vogelbusch valorization of these materials with yeast was pursued.

Brunel objectives fall into two parts: the development of design resources and strategies for Gram negative bacteria for biotech applications, and the specific objectives of developing strains for bioprocess relevant to the use of hemicellulose-derived feedstocks, and associated process development.

Design and engineering of E. coli
Strain and associated analysis resources and methods were developed for two species: K. pneumoniae and E. coli. These were progressively developed and improved over the course of the whole program, and now represent the largest co-located, sequenced, and commonly annotated strains collections for both species. They also have unique underpinning bioinformatics resources for behavioural determinant and exploitable bioPart discovery, including a fully operational ‘genome wide association study’-like analysis that can address all coding sequences, and can find exploitable components with single nucleotide resolution. To our knowledge, it is the only resource of its type for bacteria. What has been learned in this project has been applied through a subsequent separate program to a further set of strain collections for species of high priority for microbial biotechnology and synthetic biology, in collaboration and with support from SynbiCITE, the UK’s synthetic biology accelerator. And, discoveries from the Valor Plus program in this regard are being made more widely available through this route, as well as through a spin-out founded upon these resources and design capabilities.

Extensive analyses of the determinants behaviours relevant to the use of C5-rich hemicellulose-derived feedstocks and target product generation were addressed, in both starting species. These included: xylose and arabinose use, furfural (feedstock inhibitor) tolerance, native fermentation product generation (in Klebsiella only), and candidate product tolerance (isobutanol, 2,3-butanediol, ethanol). At later stages additional studies were performed addressing a novel carbohydrate nutrient supplement strategy for process development, suggested by the outcomes of early-stage comparative genomics studies, and also of 1,3-propandiol.

Based upon holistic analysis and early engineering experience, E. coli was selected as the primary species for development, and product tolerance was identified as the most important phenotype to develop to achieve advances in the field. Mutants were screened and developed based upon two evolutionary genomics strategies: the identification of markers from analysis of the diverse strain collections, and from the evolution of strains under product selection (initially isobutanol, subsequently 2,3-butanediol). An unmarked strain with substantially increased tolerance to multiple products has been generated, with transferrable increased tolerance for isobutanol, ethanol, 2,3-butanediol, and 1,3-propanediol – which for the latter three include concentrations that would be economically viable to extract.

Use of a novel process development strategy based upon insights from comparative genomics, and further screening have revealed the presence of native use of C5 sugars in E. coli that rival and exceed those of K. pneumoniae. And, two super-performer strains for future investigation and exploitation. In addition, a process initially developed in WP8 has been used as an additional route to C5 feedstock use, for the generation of nanocellulose as an additional potential value product.

Evolution under selection methods and analysis resources have been developed and used in this study that provide a platform for a range of other applications within the microbial biotechnology field. They have been used to analyze collaborator data at TUM. And, they provide fundamental insights into the nature of evolutionary processes, and necessary modifications to how these experiments must be done to inform design for synthetic biology applications.

Characterization of butanol fermentation from pentose sugars
Formation of the solvents butanol, acetone, and ethanol by solventogenic Clostridia is a complicated fermentation process from a physiologic view, which is divided in acidogenic- and solventogenic growth. Most solventogenic Clostridia, like C. acetobutylicum, use D-glucose as well as pentoses such as D-xylose and L-arabinose that form a major part of hemicellulose plant material as substrates. In most solventogenic Clostridia the presence of glucose inhibits concurrent usage of the pentoses and other sugars by catabolite repression. This results in ineffective fermentation of substrates derived from plant biomass or the pentoses are not used at all. The objective of the group of Dr Armin Ehrenreich at the Technical University of Munich was to construct strains of solventogenic Clostridia that were able to ferment pentoses and other sugars concomitant to glucose in order to allow high solvent yields from hydrolysates of hemicellulose materials.

This catabolite repression was studied in two strains of solventogenic Clostridia by transcription analysis using DNA microarrays. In order to circumvent catabolite repression it was decided to obtain mutant strains defective in carbon catabolite repression. It was expected that those strains would be able to simultaneously and completely consume pentoses and hexoses in the presence of glucose. More than 110 such mutants were obtained by EMS chemical mutagenesis and by enrichment in a continuous culture followed by selection of suitable clones. In order to investigate the sugar utilization of those mutants, they were grown in serum bottle cultures on a mixture of D-glucose, D-xylose, L-arabinose and D-galactose. The fermentation profiles of the mutants were compared with the wild type and the mutants were grouped in three groups according to their sugar consumption. Group I mutants showed complete and simultaneous consumption of all four carbon sources. Group II and group III mutants showed only moderate or no improvements of sugar consumption. Genome sequencing and analysis of all characterized mutants was done in close cooperation with the group of Nigel Saunders from Brunel University. Several mutations were identified in the genome sequences of group I mutants that were potentially responsible for the carbon catabolite repression negative phenotype. They show frameshift or point mutations in the glucose- or mannose-specific phosphotransferase system (PTS). The individual mutations are located in the genes coding for EIICBA of the glucose-specific and EIIAB in the mannose-specific PTS, respectively. In some cases, frameshift mutations were discovered in genes encoding a permease (CA_C0751) and the sigma factor of SigD/WhiG family (CA_C2143), respectively.

The group I mutants utilize arabinose, xylose, and galactose simultaneous to glucose and thereby significantly increase the solvent yield on substrates containing a mixture of such sugars such as a hemicellulose hydrolysate obtained from plant materials. Until now the most effective mutant identified was C. acetobutylicum strain 2711a.

The most effective group I mutants that were further used for pilot fermentations, originate exclusively from the enrichment by continuous cultures in chemostats, while the chemical mutagenesis resulted solely in group II and III but no group I mutants. This is very important information for further isolation of improved strains and for obtaining catabolite repression deficient strains of other solventogenic Clostridia.

Characterization of ethanol fermentation from pentose sugars
Yeast strains from the Vogelbusch Cell bank were tested for their ability to convert hemicellulose hydrolysates into ethanol. Because of low ethanol yields but high yields in xylitol the research work was modified to the production of xylitol. A selection system and process for the section of the best strains was developed. In this testing scheme strains were cultivated on different sugars that are expected to be found in hemicellulose hydrolysates (xylose, arabinose, glucose, mannose and galactose). A real hemicellulose hydrolysate was produced and analyzed for sugars and inhibitory substances. Analytical procedures were developed to analyze the various substances in the feed stock and the large number of potential products such as ethanol, xylitol, arabitol, mannitol, glycerol, lactic acid,... as well as for inhibitory substances. Selection tests were carried out at different temperatures, pH values, oxygen concentrations and over long time periods. The most promising strains were then further tested with mixtures of sugars, real hemicellulose hydrolysates and different media additives (trace element solutions, macro element solutions containing phosphate and nitrogen components and vitamin mixtures). It was possible to select about 10 strains for which a fermentation process was developed. Each one of these strains has its own strengths. The data from the experiments were evaluated and used as a foundation for the work carried out in WP 4.

Characterization of single cell protein fermentation from pentose sugars
Yeast strains from the Vogelbusch Cell bank were tested for their ability to convert hemicellulose hydrolysates into single cell protein (biomass with a high protein content). A selection system and process for the section of the best strains was developed. The goal was to find strains that are capable of metabolizing all sugars that are found in hemicellulose hydrolysates without formation of side products. In these testing schemes strains were cultivated on different hemicellulose sugars: xylose, arabinose, glucose, mannose and galactose. Tests with acetic acid were carried out as well. A real hemicellulose hydrolysate was produced and analyzed for sugars and inhibitory substances. Analytical procedures were developed to analyze the various substances in the feed stock and the large number of potential products such as ethanol, xylitol, arabitol, mannitol, glycerol, lactic acid,... as well as for inhibitory substances. Selection tests were carried out at different temperatures, pH values and oxygen concentrations. As efficient biomass production requires high aeration rates tests were carried out with specially designed shaking flasks. The most promising strains were then further tested with mixtures of sugars, real hemicellulose hydrolysates and different media additives (trace element solutions, complex media additives such as peptone and yeast extract, macro element solutions containing phosphate and nitrogen components and vitamin mixtures). It was possible to select about 12 strains for which an highly aerobic fermentation process was developed. The data from the experiments were evaluated and used as a foundation for the work carried out in WP 4.

WP 4: Prototype hemicellulose processes based on results from WP3 “C5 Sugar fermentation”
Task 4.1: Prototype process for the fermentation of pentose sugars to butanol.
While the traditional fermentations with solventogenic Clostridia, producing butanol, acetone and ethanol used starch from corn as fermentation substrate, this is undesirable nowadays in order to avoid competition to food production. Thus, lignocellulosic substrates, such as straw, gained interest as substrates for solvent fermentation. Hemicellulose, which is mainly composed of D-xylose, L-arabinose and D-galactose is an important component of most lignocellulosic substrates. A major difficulty in fermenting real-world hydrolysates of hemicellulose from plant material is that they inevitably contain significant amounts of D-glucose beside the mentioned sugars. The D-glucose is a hydrolysis product from cellulose, the main component of plant cell wall. Unfortunately, glucose is a strong catabolite repressor for solventogenic Clostridia such as C. acetobutylicum, which hampers the utilization of the other sugars, resulting in their slow and incomplete usage during solvent fermentations. In work package 4.1 laboratory scale fermentations were established to study the solvent fermentation of mutants created in package 3.2 with hemicellulose hydrolysates.

The promising group I C. acetobutylicum mutant strains developed in work package 3.2 were initially used in laboratory scale fermentations on synthetic media containing glucose, xylose, arabinose and galactose. We chose to use synthetic media containing a mixture of glucose, xylose, arabinose and galactose in concentrations that were expected to occur in hemicellulose hydrolysate with the major share of xylose and arabinose, because at that project phase supply of larger quantities of hemicellulose hydrolysate was limited. Fermentation of the wild type showed a clear hierarchy in sugar utilization by C. acetobutylicum. Glucose and arabinose were consumed simultaneously, with a strong preference of glucose. Xylose was only metabolized to a certain extent after glucose was consumed, then the fermentation stopped. Galactose was not consumed at all in this medium. By contrast, for example, the group I mutant C. acetobutylicum 2711a showed simultaneous consumption of all four sugars from the beginning of the fermentation. The fermentation only stopped, when all sugars were completely consumed. Comparison of the sugar consumption rates confirmed the significantly improved utilization of xylose, arabinose and galactose. The mutant again consumed all four sugars simultaneously and produced a final butanol concentration of 185 mM (13,7 g/L), which proved that the mutants are able to ferment a mixture of carbon sources to butanol with much a higher efficiency than the wild type.

The mutants were also studied in ABE fermentations using native hemicellulose hydrolysates produced in the project. In such batch fermentations, the group I mutant 2711a grew to a slightly higher optical density than the wild type, whereas it produced significant higher amounts of solvents. Despite some difficulties in analytics due to particulate material and colorization of the medium, it could be verified that also on this substrate a 20% to 25% increased yield in butanol and acetone cold be achieved by using strain C. acetobutylicum 2711a as compared to the wild type.

Furthermore, the consequence of the addition of pure glycerol to the synthetic medium simulating hemicellulose hydrolysates was studied as well as the addition of purified glycerol obtained from project partners to the fermentation medium. In such experiments, it was observed that the addition of glycerol results in a strong increase of butanol production.

Task 4.2: Prototype process for the fermentation of pentose sugars to xylitol (before ethanol): will follow the same procedure as for development of the butanol fermentation process.

A prototype fermentation process was developed with yeast strains for the efficient conversion of hemicellulose hydrolysates into a high concentration xylitol solution (> 130 g/l). Development of downstream processing technologies was started that form the basis for a complete production process to crystalline xylitol.

The raw material for the fermentation process was a real hemicellulose hydrolysate produced from wheat straw and an artificial sugar solution resembling the real hydrolysate. The hydrolysate contained the sugars xylose, arabinose and glucose. Inhibitory substances were not detected by analytical methods, which was confirmed by the good fermentation results with the produced hydrolysate. The fermentation process itself starts from a cell bank that is transferred into a working cell bank. With the biomass from the working cell bank a pre-culture in a shaking flask is inoculated. The pre-culture from the shaking flask is used to inoculate the next larger fermenter. The main fermenter is temperature controlled (32°C) mixed and aerated (controlled aeration). Aeration is also used for mixing with a little support from an agitator. The fermentation process is started as a batch process with a high sugar concentration. The presence of glucose does not cause any problems in the xylose to xylitol conversion process but rather helps to produce sufficient biomass to run the fermentation process efficiently over long periods of several hundred hours. The main fermentation is operated as a repeated batch process. The yield depends on the way the fermentation is run and can vary between 85 and close to 100 % for the conversion of xylose into xylitol. The yield is related to the productivity, which is lower at high yields. In the downstream processing biomass can be separated easily by centrifugation or by filtration. Chromatography such as simulated moving bed technology was tested for product stream purification. Overall a stable prototype fermentation process was developed.

Task 4.3: Prototype process for the fermentation of pentose sugars to single cell protein for animal feed applications: will follow the same procedure as for development of the ethanol fermentation process.

Yeast strains, that are capable of converting hemicellulose hydrolysate sugar mixtures (xylose, arabinose and glucose) into biomass had been pre-selected prior to the development of a prototype fermentation process. A fermentation process was developed that uses complex media components such as peptone and yeast extract. A second process was developed with micro and macro element mineral salt mixtures and in certain cases vitamin addition. As raw material for the prototype fermentation processes a hemicellulose hydrolysate (real hemicellulose hydrolysate) was used that was produced in our laboratories from wheat straw by an acid treatment process. Because this “real hemicellulose hydrolysate” was not sufficient to run all fermentation trials an artificial hemicellulose hydrolysate was prepared by mixing sugars (xylose, arabinose and glucose) in the same concentration ratios as found in the “real hemicellulose hydrolysate”. Inhibitory substances were not detected by analytical methods, which was confirmed by the good fermentation results with the produced hydrolysate. Yields close to 50% were reached (dry biomass/fermented sugar) in the fermentation runs. With a measured protein content of up to 46%, the yeast that, can be produced, by fermenting hemicellulose hydrolysates based on our fermentation technology, a great animal fodder material can be manufactured. The fermentation process itself starts from a cell bank that is transferred into a working cell bank. With the biomass from the working cell bank a pre-culture in a shaking flask is inoculated. The fermentation technology applied is a batch process to start up the fermentation run in the main fermenter followed by a continuous fermentation with high feed rates. The fermenters are temperature controlled at roughly 30 – 34°C and highly aerated. In certain cases arabinose is accumulating in the fermentation broth but that could be reduced in a second stage final fermentation following the main fermenter. The fermentation process is a continuous fermentation. Biomass separation by centrifugation was tested successfully. With the fermentation technology developed in this project it is possible to convert hemicellulose hydrolysates into a high value animal feed product that is competitive with materials such as soy meal or soy protein feeding mixtures.

For the production of biomass (single cell protein) the fermentation process can be integrated with the exiting know-how that Vogelbusch has in the field of single cell protein production. Therefore, with a robust fermentation process for the conversion of hemicellulose hydrolysates, a complete process package can be assembled and offered from raw material sterilization down to packaging of the dried product.

WP5: Lignin Depolymerisation
The objectives of this work were therefore to develop a methodology for selective precipitation and filtration of lignin to achieve greater uniformity with regards to the lignin size and chemistry, thereby enabling greater control during catalytical and enzymatical depolymerisation. Additionally the economical demands for an industrial applicable process were considered.

For the simplification of the process development a commercial available lignin called ProtobindTM1000 (Green Value SA, Switzerland) was used as staring material. A solvent based extraction process was developed for the extraction of technical lignin which could be transferred in a technical scale. This extraction process was a precondition for the development of a filtration process to obtain lignin fractions with narrow and defined poly dispersity.

Different solvents were tested to achieve an extraction of the lignin molecules from the starting material ProtobindTM1000. The final selection for the solvent was achieved by consideration of the following aspects:
• Easy evaporation of the solvent
• Low viscosity of the extracts to avoid clogging in filtration membranes
• Solvent recyclability
• Uncritical handling (non-toxic, carcinogenic etc.) of the solvent.

The studies on fractionation have been performed by POLIMI in collaboration with the partners of the ValorPlus Project BIOBASIC and IFAM. The raw lignin material was subjected to successive filtrations through membranes of decreasing cut-offs. The performance of the process was assessed on the basis of the characteristics of the resulting lignin fractions, which were analysed in terms of their chemical, physical, thermal and structural properties.

By the membrane treatment the molecular weight of the obtained fractions could be adjusted very well in different type of solvents. This means that the process is very effective and the separation is quite independent of the solvent used. However, the new process in a common organic solvent seems to be more suitable according to the initial scope of this workpackage and also to the needs of the subsequent processing in other workpackage selected organic solvents increase the solubility of lignin and the obtained solutions are easier to process downstream (sodium hydroxide free solutions, solvent evaporable and recyclable, no needs of acidification and further extraction steps....).

It should be pointed out that, in the set-up of the process, the fractionation of lignin has been optimized for ProtobindTM1000 but the principle is applicable to many different lignins. The described pretreatment process is a very valuable improvement for the lignin industrial valorisation allowing the availability of well-defined fractions with tailored characteristics in view of their potential further exploitation for the development of bio-based polymers and chemicals.

Another noteworthy achievement of this workpackage was the development of an IR-spectroscopy based method for the determination of the hydroxyl value of lignins. The hydroxyl value is a measure of the content of free hydroxyl groups in a chemical substance, usually expressed in units of the mass of potassium hydroxide (KOH) in milligrams equivalent to the hydroxyl content of one gram of the chemical substance. Infrared (IR) spectroscopy is a vibrational spectroscopy and is based on the interaction of the radiation with a material.

WP6: Chemo-Enzymatic Transformation
Lignin is a bio-based aromatic amorphous polymer that acts as the natural glue providing structural integrity to plants. It is one of the most abundant biopolymers in nature just following cellulose and represents between 15 % to 40 % (w/w) of the woody plant’s dry matter. It is one of the most recalcitrant biopolymers found in the cell wall of plants due to its natural resistance to the degradation induced by chemical and biological sources. Lignin consists of a complex methoxylated phenylpropane skeleton whose structure and chemical/physical properties are strongly dependent on its natural origin and on the extraction method used to obtain it. Although lignin represents a highly abundant aromatic feedstock, thermovalorisation seems currently to be the most convenient exploitation strategy for this cheap and highly available type of biomass and up to now around 95 % of lignin produced worldwide every year is burnt for energy recovery.

However, considering its aromatic nature, other higher-added-value applications of lignin are highly desirable and are currently extensively investigated. The lack of real effective processes that would add value to the lignin processing can be mainly attributed to its chemical recalcitrance due to its structural complexicity. In fact depolymerisation which is at the base of the lignin valorisation can be realized by chemical or thermochemical methods: these processes are energy-consuming and environmentally unfriendly and generate complex mixtures of products due to the repolymerisation of small components present in the starting raw material. As a suitable alternative, biocatalytic methods have been studied because they allow the use of mild conditions and greener reagents. The most interesting routes to profitable lignin valorisation are also its use as precursor of bio-based polymeric materials or as bio-sourced platform of chemical intermediates of interest for a wide range of industrial sectors.

For these reasons the main objectives of the WP6 have been:
• to develop an enzymatic tool box for the selective transformation of lignin functionalities;
• to develop an integrated chemo-enzymatic process for the selective depolymerisation and transformation of standardised lignins to discrete families of value macromolecular and monomer product streams;
• to develop and apply a novel semi-continuous chromatography technique for separation of the resulting mixed lignin product streams;

During this project, the first important achievement has been the creation of a quite complete tool-box of enzymes that are able to undertake specific and targeted transformations of lignins within optimum ranges of temperature and pH. The enzymatic activities/specificities of the tool-box have been selected in order to collect the main selectivities on the functional groups present in the lignin backbone. The tool-box contains enzymes obtained from commercial sources and from molecular biology techniques. The biochemical characterization of all enzymes has been performed. Their industrial production has been set-up and optimized in terms of production media, physical and technical parameters, improvement of the down-stream processing in order to scale-up the production of the proteins of the tool-box. At the moment 10 laccases, 6 Manganese peroxidases, a lignin peroxidase, a chloroperoxidase, a catechol-2,3-dioxygenase, a DypB peroxidase, a Manganese superoxide dismutase, an O-demethylase LigM and the Lig DEFLG enzymatic system are the members of the tool-box.

The second part of the WP6 main research has been focused on the lignin fractionation which appeared to be the essential tool required for the direct valorisation of this highly complex three-dimensional material in the attempt to improve the control over the chemical composition and functionality of lignin as well as its physical and structural properties. In this respect, lignin fractionation approaches conventionally rely on the independent use of chemical (pH-assisted precipitation, solvent extraction) and physical (membrane-based filtration) methods, or on their sequential combination. The research performed in WP6 in collaboration with WP5 has been a deep study on the set-up and the effectiveness of membrane-assisted ultrafiltrations on the chemical/physical properties of commercial lignins. The main achievements have been the extraction, the recovery and the full characterization of each fraction coming from the fractionation processes in terms of chemical composition, molecular weight (MW) and physical properties. It should be pointed out that the determination and the possible adjustment of the MW and the composition of the fractions are fundamental for their successful valorisation as precursors of chemicals and polymeric materials.

The fractions with a higher MW have been submitted to a cascade process based on a first step of chemical degradation and then a second step based on an enzymatic treatment. The two-step process has been set-up because enzymes were ineffective if they were used directly on polymeric material. Combining a base-catalyzed chemical treatment and a biocatalytic step, a clear reduction in terms of MW of the starting material has been achieved even though the total yields in terms of mass recovery of the two-step process didn’t exceed 20 %. Further studies are still in progress for the optimization of the whole process. Moreover the possibility of functionalizing these small products coming from the partial lignin degradation is very appealing in the preparation of new composited and new polymeric materials.

The lower MW fractions coming from the fractionation processes have been submitted to preparative chromatographic separation in order to recover the small monomers which could be used in preparative organic chemistry. A new analytical HPLC protocol has been developed for the separation of the mixed lignin product streams.

Concerning the higher MW fractions coming from the fractionation, they have been tested as starting materials for Material Science. In fact, it should be noticed that at present, lignin is mostly produced as industrial residue in pulp and paper factories as well as in biorefineries. Only a little fraction is employed for commercial uses as filler, additive or dispersant in concrete, composites, binders or coatings. However, these relatively low-value niche market applications only account for a minor fraction of the current worldwide lignin production, thus highlighting the urgent need of developing new strategies for the valorization of lignin as abundant macromolecular system for the production of high value-added, sustainable materials. Unfortunately, commercial lignins are typically characterized by highly variable and often very different properties that inevitably limit their straightforward usability. Based on these considerations, the activities carried out in the WP6 aimed at the valorization of lignin as precursor of new added-value macromolecular systems. In particular WP6 achievements have been focused on the following items: a) the solvent fractionation of a commercial softwood kraft lignin; b) the development of lignin-based polyurethane coatings; c) the development of lignin nanoparticles by ultrasonication and their incorporation into waterborne polymer nanocomposites; d) the development of bio-based thermosetting polyester coatings based on functionalized kraft lignin.

WP7: Chemo-Microbial Transformation
WP 7.1 - 7.3: Chemo-Microbial Transformation of lignin extracts
• Development of a HPLC-method for the quantitative detection of monomers in lignin extracts.
• Identification of metabolic pathways of Ps. putida as gene recombinant targets for the improvement of the production of polyhydroxyalkanoates (PHA).
• Modification of Ps. putida KT2440 strains by using different gene recombinant techniques.
• Development of an integrated chemo-microbial processes for the selective depolymerization and fermentation of lignin monomers to PHA.
• Purification of PHA from the fermentation broth.

Main results
A HPLC-method was developed for the quantitative analysis of lignin extracts.

The alkaline treatment of lignin under comparably moderate conditions (4% NaOH, 80°C, 2h) provided a high number of monomers. At least 14 different monomers were detected by HPLC.

It was observed, that several of these compounds were consumed by Ps. putida cultivated on media with 20 % lignin extract. After examination of the metabolic pathways of Ps. putida two gene recombinant targets could be identified for the improvement of the PHA-production:
• deletion of the glucose dehydrogenase (gcd) gene
• overproduction of a pyruvate dehydrogenase subunit (AcoA).

Using gene recombinant techniques the knockout mutant Ps. putida KT2440 ∆gcdstrain was generated carrying a deleted glucose dehydrogenase gene.

Compared to the wild type this knockout mutant showed an increase of PHA-yield of 60 to 600 %, depending on the substrate.

The gene acoA coding for the enzyme pyruvate dehydrogenase was successfully isolated from Ps. Putida KT2440 and inserted into the expression vector pSEVA424. The resulting plasmid pSEVA424acoA was cloned into the strains Ps- putida KT2440 and Ps. putida KT2440∆gcd.

A biotechnological process for the production of PHA was developed.

The best results were achieved using a combination of alkaline lignin extract from grain stillage and crude glycerol as substrate yielding 3.7 grams PHA per liter fermentation broth for of Ps. putida KT2440 ∆gcd.

Cupriavidus necator turned out to be more effective than Ps. putida KT2440∆gcd for the production of PHAs. Using alkaline lignin extract and biodiesel waste the fed batch fermentation process gave a yield of 14 g PHA per liter fermentation broth This means that more than 60% of the dried biomass consisted of PHA.

The PHA was purified to homogeneity and used for the generation of films.

WP 7.4: Lacquer development
Target of the work package was the chemical modification of refined lignin macromolecules for the syntheses of novel coatings and adhesives. A work program for chemical modification of lignin macromolecules for the use of lignin based resins in coating primers has been carried out. Another goal was to create a Lignin-Polyester applicable for coating formulations, manufactured with an appropriate reaction rate.

The work aimed to develop a binder on the base of the renewable raw material lignin.

Main results
As raw material a commercial available lignin, called ProtobindTM1000 (Greenvalue SA, Switzerland) was used as starting material.

The Lignin was chemically modified to create new binders on epoxy-, amine- and alkyd basis. Formulation and manufacturing of a primer based on Lignin ester epoxide have been performed. In another approach lignin was used for the formulation of a polyurethane-primer as well. Additionally the kinetics of the conversion of isocyanate with the selected epoxy resin and lignin fraction by FT-IR-spectroscopy was investigated. Amino-modified lignin was used for the formulation of water based epoxy coatings. The performance of the obtained coatings was tested according different standards.

WP 7.5: Utilization of refinedLigninmacromolecules as functional additives within biopolymercomposites
Main objective of this task was the development of surface modified lignin macromolecules to improve PLA material performance. In order to achieve this target, three main approaches have been developed to obtain surface modified lignin macromolecules.

Main results
For film processing , at least few hundreds of grams of modified lignin were required. To obtain the required amount of each component involved in the process, reaction conditions and yield of modification 2 (lignin grafting) and modification 3 (grafting of PLA onto already modified lignin macromolecules) were studied and upscaled. PLA based lignin compounds processing was based on the use of a laboratory twin screw extruder with a flat film die. Films were obtained with an average thickness around 55 micron. Mechanical properties showed an increase of around 10% in Young Modulus and an elongation at break of 90% compared to neat PLA. Generally, increasing wall thickness reduces deflection during impact and increases the energy required to produce failure. In simple, flat-wall sections, each 10% increase in wall thickness provides approximately a 30% increase in stiffness. It is considered that potential decrease associated to use of lignin composites can be around 20%. The thermal properties of the composites are not significantly affected by the introduction of lignin, showing similar values than pure PLA. Barrier properties were not significantly improved. Therefore, main application of the composites can be on short shelf life applications.

WP8: Glycerol Valorisation
Part 1 - CARTIF

Objective 8.1: Crude glycerol characterisation, specification of microbe engineering strategy and identification of microbe strains

Crude glycerol from biodiesel production using soybean oil as raw material was treated in order to characterize its composition. Several analytical techniques including gas chromatography, inductively couple plasma or thermogravimetry were used to obtain a complete physical and chemical compositional profile of the crude glycerol samples. Furthermore, in order to adjust some parameters such as pH value or soap content and fatty matter of the crude glycerol, it was developed a simple methodology based on the addition of 85 % orthophosphoric acid.

As a general strategy to valorise the pre-treated glycerol, different lactic acid bacteria (LAB) strains were selected based upon a comprehensive comparative genomics analysis (RADE:CBG) of phenotypic data such as growth in pre-treated glycerol and lactic acid production.

Objective 8.2: Engineering of microbe strains using RADE and characterisation of glycerol fermentation processes

A screening of bacterial strains from a range of food products (raw milk, yoghourt, kefir, whey, cheese...) was carried out in order to find some LAB able to metabolize the pre-treated glycerol as main carbon and energy source and to produce valuable organic acids such as lactic acid. As a result, 221 LAB strains were isolated and identified by 16S rRNA gene sequencing analysis. According to fermentation trials, strains which showed the best lactic acid producing potential were Lactobacillus paracasei 10q and Lactobacillus kefiri 50k, but the intra-clonal analysis methodology applied to these species was not sufficiently diverse for the standard RADE:CBG analysis. Due to the impossibility of obtaining enough genetic diversity of unrelated LAB species, it was decided to continue the ongoing research of lactate production process using the Lb. paracasei 10q strain (best producer of lactic acid) without additional genetic engineering modifications.

Objective 8.3: Identification and understanding of critical glycerol fermentation parameters within an experimental prototype system

In order to improve the conversion of pre-treated glycerol into lactic acid, the optimal conditions for lactic acid fermentation were investigated by performing a factorial experimental design. It was determined that culture medium, temperature and pH value were the most important fermentation parameters to control the process. Furthermore, a procedure for the analysis of fermentation metabolites and by-products was developed using the HPLC methodology.

Objective 8.4: Final optimised glycerol fermentation system and validation of the resulting product streams

As a final step, it was designed an experimental prototype reactor configuration for the fermentation process. The prototype reactor was successfully tested at laboratory level but lactic acid production could not be further improved, confirming the low concentration achieved during the initial fermentation trials. A positive aspect was the total consumption of pre-treated glycerol present in culture medium and the high levels of bacterial biomass obtained, which indicates that Lb. paracasei 10q strain is able to metabolize the pre-treated glycerol from the biodiesel production process and to grow using this substrate as sole carbon source. This result could have an alternative application, since Lb. paracasei is a probiotic strain widely used in dairy industry.

Part 2 - UBRUN
The starting objectives in this work package were to explore Gram-negatives as potential users of glycerol-rich biodiesel waste-derived feedstocks for process development and value product generation, and to provide supportive genomics to CARTIF for a strain and target of their selection for development. This changed over the course of the program, because processed waste was not available until later than expected, it was found to differ substantially from simulated feedstocks, and to have inconsistent properties; and CARTIF chose a target species with more than one product, which required a different approach. To these starting objectives, and informed by earlier work, two additional strategies were developed: a nutritional supplement strategy for using glycerol-based feedstocks; and a novel strategy for the conversion of glycerol-rich biodiesel waste to a value product.

The substantial variation in feedstocks, from the simulated (sigma pure glycerol-based) and waste-derived processes showed that E. coli is a good conversion platform, but that the best strains and strain-optimization pathways were highly dependent upon the specific feedstock. And, as such any such strategy based upon a feedstock other than that to be used industrially would be redundant, or even counter-productive. This was therefore abandoned, especially after it was discovered at a late stage that the processed waste provided with generated with a process that was not economically or industrially viable.

For strain development purposes using glycerol-rich feedstocks, it was decided that 1,3-propanediol is a good target product using Gram negative chassis-based / E. coli systems. This product was therefore added to the product tolerance research being developed in Work Package 3. The end result of which is an unmarked engineered strain with substantially increased propanediol tolerance, with good and better than unselected growth of the wild-type strain at up to 9% propanediol, which is a substantial increase and commercially relevant; and it is likely that this can be improved upon further.

The results of the comparative genomics analysis of growth using (pure) glycerol as a feedstock platform was used to make predictions as to likely useful process developments. From this, a new strategy has been developed that could enable the use of predominantly glycerol-based feedstocks in existing E. coli-based biotechnology and industrial applications.

CARTIF selected a Lactobacillus-based strategy, for which around 200 genomes were sequenced, assembled, annotated and analyzed. This required novel genome-based speciation tools to be developed (partly to resolve mixed cultures, partly to address misleading rRNA typing information). This new tool will be useful and made available for whole genome-based speciation in the near future, which will be useful for a range of biotechnology and food-based users. Extensive strain comparisons were performed for use of glycerol, growth rate, and product generation. Best performer and bioPart containing strain were identified in this process, and subsequently pursued by CARTIF.

A new entirely bio-based process that uses raw untreated glycerol waste was developed to overcome issues of the costs and inconsistent outputs of pre-processing. A feature of this is that it uses a microbial community to avoid the necessity for nutritional supplement such as yeast extract (or similar) making it commercially and ecologically viable solution. The process generates nanocelllose, which has been confirmed and shown to have good and better features than nanocellulose derived from commercial solvent extraction processes from wood. The process has been developed and improved with process efficiency increases of over 250% using evolutionary strategies. And, the community has been characterized using metagenomics strategies. This prototype process is the focus of intended future work and exploitation through a new Brunel spin-out, Syngenious.

WP9: Scale-up and LCA
Several activities were covered in WP9. All these activities covered the three main value chains developed in the project: (a) Hemicellulose value chain; (b) Lignin value chain and; (c) Glycerol value chain. The work within these value chains covered four common topics:

1) Scale-up and integration of the ValorPlus processes within existing and future biorefineries: Here it was designed the basic design and set-up for the scale-up of both the hemicellulose and lignin value chains based on the lab-scale results from the related WP’s. Specifically, the flows were calculated, and a fixed daily production was established for the calculation of tank and reactor volumes as well as the auxiliary equipment (pumps, storage tanks, agitation, filtration systems) across the entire process. Different size volumes were defined, and in case of differences between the flows from enzymatic hydrolysis of hemicellulose/depolymerisation of lignin, solutions like the use of storage tanks and phase-shift reactors in parallel were suggested. In the specific case of glycerol value chain, the scale-up was carried out by using ASPEN Plus chemical engineering simulation software, being the entire process calculated and demonstrated the viability through OPEX and CAPEX calculation. The simulations delivered positive net cash flow, so that the glycerol fermentation process defined is viable from an economical and a technical point of view.

2) Road mapping: Road mapping was made through a series of meetings with project partners and stakeholders in order to extract the inventory data for the LCA, set the basic conditions in an industrial environment and the identification of the potential main products. This work included the participation in workshops. The workshop discussions covered the issues related to the higher costs expected for bio-based materials, even though it was not expected a concurrency between the agro-based origin of the materials used in ValorPlus and food production.

3) Life cycle assessment (LCA): A full LCA was carried out in order to estimate the potential environmental impacts related to each one of the three value chains suggested in the project. The LCA analysis covered all the impact categories included in the Life Cycle Impact Assessment (LCIA) method ILCD 2011 Midpoint+ which covered different harmful effects in the environment like Climate Change, Ozone Depletion, Eutrophication, Human Toxicity or the Use of Mineral and Fossil Resources. This LCA was conducted based on the Harmonised Guidelines for LCA of bio-refineries coming from SupraBio, BioCore and EuroBioRef EU projects and the new European Standard EN 16760:2015, although the functional unit and the system boundaries were customized for the specific case of ValorPlus. Therefore, the functional unit was 1 ton of agricultural feedstock (dry basis) leaving the field, while the analysis was carried out in a cradle-to-gate perspective, excluding the distillation/rectification of the products from the fermentation broth in the hemicellulose and glycerol value chains. In the case of the lignin value chain, the extraction and purification of PHA from GMO P. putida fermentation was considered. Main results achieved through this LCA analysis were:

a. Hemicellulose value chain:
i. Fermentation processes do not show the biggest contribution to environmental impact, except in the case of the aerobic fermentation by yeast to produce single cell proteins (SCP).
ii. Peptone plays a relevant role to the environmental impact of the fermentations carried out with yeast.
iii. The impacts of cellulase used during the enzymatic hydrolysis of hemicelluloses are not high, while the inorganic salts used in the in the enzymatic hydrolysis contribute significantly to environmental impact.

b. Lignin value chain:
i. Most of the impacts are concentrated in the downstream steps after the PHA production by fermentation with P. putida of depolymerised lignin, mainly because of the use of chloroform as solvent, although a chloroform recovery system will reduce such impacts for sure.
ii. The impacts of the agricultural feedstock for the production lignin stillage and bioethanol does not have a relevant impact except to land use impact.
iii. The chemical depolymerisation and GMO microbial fermentation are not relevant in terms of environmental impact in most of the impact categories

c. Glycerol value chain
i. Crude glycerol must be pre-treated before to proceed with the lactic acid fermentation. The process can be safely carried out with sulphuric acid. This reactant does not influence the conversion of glycerol into fermentation products like lactic acid
ii. The impacts of biodiesel/glycerol production are by far the main contribution to impact, while impacts from lactic acid bacteria fermentation does not exceed 30% in most cases
iii. Based on the outcomes of the LCA, the environmental impacts of a fermentation unit for glycerol process are lower than expected in principle.

4) Policy Review and business plan: A policy review and a business plan was also defined for the potential products to be obtained from the above-mentioned value chains. Several key products were found to be further developed for a proper business around the use of hemicellulose, lignin and glycerol:
a. Production of chemical building blocks: (i) Xylitol from hemicellulose; (ii)1,3-PDO from glycerol
b. Production of advanced bio-fuels: Bio-butanol from hemicellulose
c. Bio-based plastics: (i) PHA from lignin; (ii) PLA from glycerol
d. Pre-biotics: Oligosaccharides from hemicellulose
e. Coatings and adhesives from lignin

Even though it was intended to carry out the policy review analysis based on the type products, this approach was not further possible because of the lack of specific policies and legislation for most of the above-mentioned products. Therefore, a general analysis of policies about biorefinery was made. The main conclusion was that investment and setting a plan for incentives are an essential part to promote the use of biomass feedstocks and the use of industrial biotechnology. The removal of trade barriers and boost the use of sustainable products from industrial biotechnology are essential, mainly in low income Countries. Providing measures for a constant supply and quality of bio-based feedstocks is also necessary for the development of integrated biorefineries based on specialty chemicals and not only to fuels.

WP10: Demonstration
This document describes the demonstration activities within Valorplus project. It describes the demonstration activities, achievements and demonstrators obtained.

The Valor-Plus project supports the realization of sustainable, economically viable closed loop integrated biorefineries through the development of new knowledge, (bio-) technologies and products that enable valorization of key biorefinery by-products. One of the main areas of the project is the demonstration of the technological and economic potential for integration and scale-up within existing and future biorefinery value chains. This point includes the demonstration of component technologies, focused biodiesel refinery case study, roadmaps for technology and product stream integration, and a full life cycle assessment.

The Demonstration Plan is intended to fully document the plans for demonstrating and validating the performance of the proposed technology, showing specifically some examples at demonstration sites. The main objective of the Demonstration Plan is to fully document the intended actions for establishing or qualifying the alternative technology at the demonstration sites and for evaluating the technology in “real” testing. This may include the application of the technology in an operational environment or manufacturing facility and/or the field testing of the technology. The results obtained should provide information on the expected operational performance and cost of the technology.

As there is a natural difference in industrial and academic intentions regarding the demonstration and dissemination of results (in general regarding the grade of detail showed) the plan considers this topic and separates the demonstration activities into “educational” and “industrial/media” activities, where the approach was more technical or more general depending of target publics.

The Demonstration work package created a wide range of technology and product demonstrators which are the frame of several demonstration events. In summary, during Valorplus prohect life, a series of DEMO activities were generated such as: 4 demonstration days, 48 paper “Technology” demonstrators, 5 hard copy “Technology” demonstrators, 6 paper “Products” demonstrators, 6 hard copy “Products” demonstrators, 2 permanent demonstration areas, 1 eventually demonstration area and 1 joint video.

Parts of the demonstrators were used in a wide range of activities including presentations on conferences and fairs (48), published articles (15) and in customer-based meetings.

Potential Impact:
Over the next years, global population growth is expected to push the population of the world up to a total of 9 billion people in 2050. Such an increase, along with the rapid depletion of many resources, external energy dependence compounded by price instability, increasing pressures on the environment, as well as climate change, are all factors that make it necessary for Europe to make radical changes in how it produces, consumes, processes, stores, recycles and uses biological resources. Growing concerns about the need to reduce dependence on fossil carbon, the need to use resources more efficiently and the importance of promoting a transition towards a renewable bio-based economy has been increasingly recognized in recent years.

By making better use of biological raw material, by-products and wastes (e.g. forestry residues, food waste), the bio-based economy will constitute an important part in the process for the further stimulation and growth of the society. Such a bio-based economy would draw on locally produced biomass to produce a variety of outputs, including chemicals and fuels that are currently still largely produced from imported fossil resources, thereby creating local jobs and growth while reducing the environmental impact of these industries.

The application of industrial biotechnology (IB) has become a key driver behind the development of new bio-based products, as it enables a smart use of microorganisms and enzymes for the sustainable processing and production of a wide variety of chemicals, compounds, materials and fuels, which are then applied across the manufacturing sector, including chemicals, pharmaceuticals, food and feed, paper and pulp, textiles, energy, materials and polymers. It helps for the replacing of petro-based products and processes, but also leads to the development of new ones, while reducing the use of crucial inputs like energy, water or chemicals in production processes. Consequently, modern biotechnology applications reduce greenhouse gas emissions, waste generation and the use of non-renewable resources.

Biomass is one of the few resources that has the potential to meet the challenges of sustainable and green energy systems. With abundant resources of renewable biomass available globally, biorefineries have been recognised as a long term solution to Europe’s resource needs whilst combating the effects of climate change by unlocking the potential to refine and utilize the complexity of biomass feedstock, by its conversion into a wide range of bio-based products, such as fuels, chemicals, power and heat, materials, food and feed. The development of this industry opens important opportunities for many countries in a context of sustainability and competitiveness.

Nevertheless, biorefineries are a capital-intensive project, and when they are based on just one conversion technology, or target the production of a single product stream using a single fraction of the biomass, they increase the cost of outputs (or products) generated from such biorefineries. The focus on only a single product stream and fraction of the biomass leads to a number of important limitations like the production of large volumes of waste by-products; poor commercial competitiveness; request of feedstock crops rich in the target sugar/oil fraction (which are nutrient intensive and compete with food crops); or reduction of biodiversity due to crops needed.

Hence, several conversion technologies need to be combined together to reduce the overall cost, as well as to have more flexibility in product generation (integrated biorefinery). An integrated biorefinery is designed to maximize the valued extractible while minimizing the waste streams, enabling full use of the biomass (zero waste) and the optimal use of available resources, improving process efficiency (multiple product stream processes), thereby generating the highest value return and increasing the commercial competitiveness and profitability; thereby enabling greater flexibility in platform configuration, customisation towards end-user requirements and access to new niche markets.

The main objective of the VALORPLUS project is the development of integrated closed loop biorefineries that ensure their sustainability and economical viability through a complete use of biomass, minimizing waste, and generating the greatest possible added value from the available resources. To this end, the research and development carried out in the course of the project is aimed at developing quality control procedures for the reliable and consistent recuperation, treatment and revalorisation of hemicellulose fibres, minimally degraded lignin macromolecules and raw crude glycerol compounds. The applications of these processes allows to create a wide range of subproducts with potential uses across a variety of applications, ranging from biofuels to the manufacture of prebiotics, bioplastics, biomaterials and coatings (prebiotic, additives for polymer composites, adhesives and coatings, fuel additives and other chemicals of interest such as ethanol, butanol, 1,3-PDO, etc.).

The new biorefinery concept which provides the framework of this project marks a clear step forward in that it overcomes problems arising from the generation of residues by giving them new value. This is how a significant increase in profitability and competitiveness over petrochemical equivalents will be achieved – due to a greater efficiency derived from generating multiproducts; a reduction in dependency on food crops by using a broader range of biomass resources (agricultural and forestry residues); and also by creating value in something which initially lacked any such value.

The project targets the generation of bulk and high value product streams from the byproducts lignin, hemicellulose and glycerol by developing new science and technologies, and utilizing a platform of integrated technologies. The platform technologies will enable the optimum value to be achieved for each byproduct stream whilst ensuring cost-effective and complete utilization of available resources. The project therefore enables the realization of sustainable, economically viable closed-loop integrated biorefineries.

Positive economic impact on the Bio Sector
The project will have an important impact on the assimilation of the newly developed technologies. The impacts can be summed up as follows: decreasing the dependency on oil and fossil based products; the adoption of more sustainable practices in the agricultural economies; improving biodiversity through the new varieties of lignocellulose crops; tailored solutions for industries which produce new bio-based products; rural development; and the development of other industries due to the links and overlaps between companies, products and services. Availability of cost-competitive biomass conversion technologies plays crucial role for successful realization of biorefinery for sustainable production of fuels and organic chemicals from biomass.

To ensure effective technology & knowledge transfer activities, effective communication and dissemination tools were defined. The dissemination actions aimed to keep all the stakeholders fully informed about the project status, planning, results and main achievements in order to obtain transparency for all partners and actors interested.

To achieve this goal, a variety of activities and materials have been prepared and carried out for the four years of the project to reinforce the effective communication and dissemination of the project outcomes, and to show to interested stakeholders the evolving conditions in which the project has being developed. Communication and dissemination activities have been closely related with DEMO activities, reinforcing the impact on interesting target groups and stakeholders.

From the dissemination point of view, it has been fundamental to increase the visibility of the project, stimulating the interaction between partners, improving internal communication, and working towards the outreach of most promising results derived from the project.

All the information generated in the VALORPLUS project has been circulated among all the partners of the consortium. It offered the opportunity to have the greatest possible cooperation and improve the transfer of knowhow. Last results and relevant information have been elaborated and distributed amongst them as the project moved towards the development of close loop biorefinery technologies and processes.

The general objectives of the Dissemination and Communication Plan are:
• To optimise the flow of information to promote efficient communication and dialogue between the institutions participating in the project and other possible interested parts or end users to establish and maintain mechanisms for effective exploitation.
• To set up a stable and continuous dialogue within the project potential stakeholders and key beneficiaries to encourage the interactions/networking.
• To report and communicate the results obtained to public and private entities and European institutions from national and European levels that might be interested in the project.
• To ensure that information is shared with appropriate audiences on a timely basis and by the most effective means.

The specific objectives of outreach and external communication are:
• To inform the target audience about the Project Idea: objectives; reason for its creation; composition of the consortium; results; etc.
• To disseminate the progress and results achieved during the project.
• To provide documentation and reference material for future work or collaborations.
• To disseminate and transfer new findings or reference material for policy makers at regional, national and European levels.
• To engage in effective, transparent and understandable communication with the society in general, focusing on the project ideas and benefices: sustainability, innovation, bioeconomy, viable exploitation, scientific potential, the new biorefinery concept, etc.

The project has to reach different kinds of audiences. The types of public we have found are:
• Internal direct audience: Consortium partners and stakeholders responsible for management and coordination within the European Commission.
• Direct external beneficiaries: Innovative and / or technology-based companies and SMEs, intermediate bodies such as research institutes, universities, etc. and specialised companies participating in innovation, Biotech clusters, Associations and Technology Platforms.
• End users: Suppliers of raw materials and intermediates (Farmers, Foresters, Local cooperatives, Large producers, Waste managers, Pulp mills, companies of the agroindustry sector), producers of bio-based (end-) products (Process designers, Catalysts and enzymes producers, Biorefining companies), chemical companies, Automotive companies; Construction companies; Chemical companies; Energy companies; Packaging companies; Polymers and composites producers, Food companies; Animal feed producers; Sweeteners and food additives producers, etc.
• General Audience: Agencies and public bodies at European level, Public & Business-Interest NGO’s, Government, Society in general.

The strategy of direct beneficiaries and other recipients has included the following lines of action and dissemination tools:
• Corporate Image: Creation of a logo and corporate image, Project Website.Link to VALOR PLUS project in partners´ website, Participation in Social Networks
• Promotional Material: Creating brochures, banners or project overviews disseminating information on project objectives and main lines, progress reports, develop promotional material for seminars, press conferences, meetings, workshops and conference tables, develop demo materials to show them in seminars, meetings, workshops and conference.
• Public acts and Events: Meetings, Kick off and Final Project Conference, Demo events and workshops
• Periodic targeted campaigns: Press Releases / Articles, News and events on the website of the project, Products Application notes
• Science and society: Scientific papers and posters, Video uploaded in ValorPlus website and YouTube channel, News and events addressed to link science and society

• Logo: A specific project identity was created in order to reinforce and establish a project’s external image via a logo which reflects the VALOR PLUS acronym as well as the background and basis for the project in an original way. The logo was selected because of its clear and direct design, and the general concept that the plus sign represents.

• Project website: A website was launched during first months of the project where main information of the project is summarised, and stakeholders, interested entities and clusters can receive key information about project evolution and results obtained. The aim of the VALOR PLUS webpage is to offer information about the project, including objectives, structure and partners, to everyone. The project website is a central element that serves not only to provide direct information, but also as a tool for the dissemination and delivery of other materials, such as reference material, publications, brochures and logo. Regarding the website impact, it keeps awaking the interest of the public since we have obtained a total of 20.895 page views, with 4.018 sessions, with a rate of 69,61% for new visitors.

• Social networks: The different ValorPlus social networks count actually with more than 300 followers and more than 1.000 impacts (Twitter: 1,248 Tweets and 140 followers; LinkedIn: 103 members; Facebook: 59 followers and 60 likes).Through them, most important news about economy and biorefinery processes have been showed, stimulating the public reflection and informing about opportunities, challenges and European measures to improve and promote this area and related projects, identifying main actors.

• Brochure/leaflets: We have elaborated 1.000 brochures which have been delivered in the different meetings and conferences where partners have participated.

• Two banners, case study and progress report were developed, in which major points were defined and project achievements were summarised and presented in the principal events.

• Demo materials: they have been exposed in panels in Universities and in demo events. In order to show methodology and the results of the project in a DEMO plant to international journalists, students and researchers or stakeholders. The idea is to showcase through informing panels and DEMO materials the technologies developed within VALORPLUS and the products obtained, and how they could be included in the biorefinery value chain for an optimum use of the biomass

This dissemination material has been shown and shared in more than 50 events.

• Videoclip: For the dissemination of the project, a video was created in order to create awareness between the scientific community, and the biotech and biorefinery sector, making it also attractive for the general public.

• General presentation: Very short summary of the scope, main results and consortium participants was developed to be used for general VALOR PLUS dissemination.

By contributing to scientific Conferences & Events, we aim to present main objectives and outcomes of the project, to stimulate the interest of the general and industrial public.

A total of 13 meetings have been carried out during project life where major inputs and results were shared with project consortium. Several additional meeting were carried out to define and explain parameters and information required within WP9.

The Demonstration work package created a wide range of technology and product demonstrators which are the frame of several demonstration events. In summary, during Valorplus prohect life, a series of DEMO activities were generated such as: 4 demonstration days, 48 paper “Technology” demonstrators, 5 hard copy “Technology” demonstrators, 6 paper “Products” demonstrators, 6 hard copy “Products” demonstrators, 2 permanent demonstration areas, 1 eventually demonstration area and 1 joint video.

Events assisted: project consortium attended more than 75 events were they have shared promotional material or presented the VALORPLUS project. Here we present those were AVLORPLUS consortium participated actively (oral presentations or posters).

• Posters: 42 posters have been prepared with the most important information of the project and were exposed in 18 events.

• Press releases: 4 press releases have been launched with more than 100 appearances in media, highlighting the beginning and end of the project major issues.

• Scientific publications: A total of 25 publications in specialized / scientific magazines have been launched during project lifetime and five publication more are in preparation.

Partners have presented and shared the project info materials at several bioeconomy events and workshops, where the attendees and invited speakers came from different areas and fields not directly related with a scientific background, ensuring the dissemination to a broad range of stakeholders, such as farmers, politicians, industry, NGOs, etc. Examples of these kind of events are:
• The Spanish Conference on Bioeconomy which took place in the Toledo Congress Centre on the 26th of September 2013 during the European Biotech week.
• Agrobiotech Innovation in Ourense, Spain on 26 November 2015 where ASEBIO presented the project to European agrifood sector.
• VALORPLUS project was presented during a session dedicated to the bioeconomy in BIOSPAIN 2016 (“Bioeconomy policy: building Europe”). VALORPLUS project was presented as a case of success.
• The 8th of November ASEBIO, ASEBIO organised with the rest of Valor Plus partners the Demo event in the CLAMBER R&D Biorefinery plant of Puertollano. The main objective of this event was to show methodology and the results of the project in a DEMO plant to international journalists, professionals, students and researchers or any other stakeholders.

To give a proper and significant relevance and visibility at local and European level, scientific articles and press releases have been published in general media and scientific or specialised magazines. This has given more visibility to the project, acting as external communication of the project.

Thesis: This kind of action not only strengthens relations with the university but also promotes employment and enriches the educational programs working with a real project with companies. Due to the researching of the project, 22 trainees have been recruited.

• Quality control procedures for the existing extrusion fractionation process enabling recovery of minimally degraded hemicellulose suitable for effective and efficient downstream treatments and for provision of reliable and consistent external sources of hemicellulose.
• Patent in process about new and advanced method of continuous processing of natural fibres for Hemicellulose extraction

• Commercial exploitation of R&D results. Licence on obtained modified hemicellulases: New Hemicellulase (xylanases) for the degradation of polysaccharides of lignocellulosic biomass (ASA +TUM)
• Publications of research for the general advancement of knowledge regarding the development of enzymes for the controlled hydrolysis of hemicellulose to oligomers, building block sugars and organic acids (TUM). Development of a broad enzyme toolbox that can be used to completely degrade hemicellulose to monosaccharides.
• Commercial exploitation of R&D results. A fermentation process (production technology) for the conversion of hemicellulose sugars into biomass (single cell protein). (VOGEL)

Chemo-enzymatic depolymerisation
• Publications for the General advancement of knowledge (POLIMI) Chemo-Enzymatic Transformation processes of Lignin did not obtained good results for exploitation.
• Commercial exploitation of R&D results: Possible licence on obtained modified oxidative enzymes and enzyme tool box (hemicellulose and lignin). Enzymes for the conversion of lignin compounds (POLIMI + ASA)

Chemo-catalytic depolymerisation
• Commercial exploitation of R&D results: Patent for “designed and developed catalysts for depolymerization lignin” (Spain, Europe and USA). (ABENGOA). ES 2642143 A1: “A catalytic process for the depolymerization of lignin”, Spanish Patent Office (Spanish Patent Application Number P201630455). 2015. Application date 12/04/2016. PCT/ES2017/070233( Application date 12/04/2017): A catalytic process for the depolymerization of lignin
• Chemo-microbial process for the conversion of lignin-based biomass to polyhydroxyalkanoates (PHA). Exploitation of R&D results via standards. Publication, presentation on fairs, application of a subsequent R&D project. (ASA)

Possible patent for “modified strains capable to grow and transform lignin”.(ASA)
Possibilities for exploitation for nano-sized lignin particles (IFAM)
Possible exploitation of: Lignin composites based biomaterials and coatings, and commercial chemically modified lignin. (ITENE, IFAM, POLIMI)

• Commercial exploitation of R&D results: Glycerol-rich waste conversion to products and raw material using a selection-enhanced co-culture (BRUNEL). Culture / strains usable under licence directly. Process patents can be pursued for licensing and development of both their 'co-culture generated new feedstock' and into the process optimizations for the use of glycerol in fermentation. Intended to be part of IP and resources for Syngenious / SynbiSTRAIN spin-out. Aiming for by end of 2017 or later based upon business planning and fund raising.

• Commercial exploitation of R&D results: Opticoli v1 STARIN PATENT. A transferrable solvent / product tolerant E. coli lacking membrane disruption and metabolic pathway modifications. Strain patenting with the design-based double mutation for single product, transferrable product, and general increased fitness in process-relevant culture conditions (BRUNEL). Strain usable under licence directly. Patents or other IPR exploitation (licenses): Designed / non-naturally occuring mutations / combinations to be protected by patent through Brunel.

• Commercial exploitation of R&D results: Strain collections. These include E. coli, K. pneumoniae, and Lactobacillus. Plus supporting analytical tools and resources refined during the project. Strain, bioPart, and design resources for biotechnology and synthetic biology, applicable to strain design and process development, and contract research services. Key resources protected by limited distribution, and trade secret protections.

• Publications for the General advancement of knowledge about the characterisation and pretreatment of crude glycerol from soybean oil (CARTIF). Definition of the quality control methods of crude glycerol. Strategy for processing the crude glycerol from soybean oil based on adding phosphoric acid and followed by a centrifugation step. As a result, three fractions are formed: fatty matter (26,11 %), the soluble phase with glycerol (62,34 %) and a pellet with soaps (11,55 %). Characterisation and analysis of the crude glycerol composition: methodology to characterise more than 25 parameters of the crude glycerol.

• Publications for the General advancement of knowledge about the screening and identification of bacterial strains from raw "lactic" material for the fermentation of processed glycerol. (CARTIF)

• Publication for the General advancement of knowledge on the determinants of glycerol-based culture of E. coli; including description of the features of 2 ‘high performer’ naturally occurring strains of E. coli (BRUNEL)

• Publications for the General advancement of knowledge about Characterisation of organic compounds produced during the fermentation of processed glycerol (CARTIF): Design a procedure for the analysis of the compounds produced during the fermentation process. Creation of a ‘design of experiments’ for the optimization of the fermentation conditions. Optimization of the bacterial strains and fermentation parameters: the Lactobacillus paracasei 10q strain was the best producer of lactic acid using glycerol as a sole carbon source. The optimal temperature and pH values are 37º and 5.6 respectively.

• SynbiSTRAIN, strain and natural bioPart discovery resources of exploitable bacterial species for synthetic biologya Brunel spin-out

• Spin off: Syngenious is a translatable knowledge-discovery enterprise with a primary emphasis on bacterial systems and the identification of exploitable strains, bioParts, and biomarkers relevant to strain selection and discovery, synthetic biology, and diagnostics. Key components of the Syngenious solution are the diverse sequenced collections of selected exploitable bacterial species: the SynbiSTRAIN collections and novel informatics-based interrogation of genome sequences for discovery genomics for: Strain selection, bioPart discovery, biomarker discovery

• Process patent (pending sequence definition of culture required) for the supplement and pre-treatment free conversion of waste to product, and to second-phase feedstock.

• Publications for the general advancement of knowledge: Of RADE strategies for analysis for ‘system-design’ using K. pneumoniae as an example of the approach, tools, and resources. Of RADE:SREP as a next generation ALE methodology, and promoting the analysis methods of Syngenious. Of combined RADE:SREP and RADE:CBG for tolerance engineering (once patent protection addressed). Of the process identifying tolerance to ethanol using RADE:CBG identifying the role of polysaccharide production / island.

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