Final Report Summary - WASTE2GO (Development and verification of an innovative full life sustainable approach to the valorisation of municipal solid waste into industrial feedstocks.)
Executive Summary:
Each year, over 180 million tonnes of Municipal Solid Waste (MSW) are discarded in the EU, which is in excess of 1kg per citizen every day. Technologies such as incineration, anaerobic digestion and composting all have a role to play in the processing of this waste but these technologies fail to exploit the chemical value contained in this material. This is where Waste2Go comes in.
Waste2Go (www.waste2go.eu) is an EU funded research and development project that is developing technologies to improve waste management and increase value –social, economic and environmental. Its main objective is the innovative transformation of the biogenic fraction of MSW, which accounts for over 55%, into chemical with an economic value greater than its use as an energy source. During the 3-year project the primary focus was on transforming specific organic waste streams such as waste paper, dry mixed recycling and black bin bag waste to useful chemical feedstocks. Once various strains of microorganisms were developed at the Norwegian University of Life Sciences for producing cellulose degrading enzymes research was shifted to the UK, where CPI developed small-scale industrially-relevant processes for cell growth and enzyme production. With knowledge gained from this work, the production processes were then successfully run at 750L scale, yielding sufficient stocks of six key enzymes to enable the project to use them in trials for the conversion of biogenic waste cellulose to valuable chemicals. In parallel a bespoke mixing vessel was also developed which enabled the project to process a range of waste derived substrates at high solids loadings in excess of 20% dry weight (w/v) which would not have been feasible in conventional stirred tank reactors. This engineering feat has expedited the scale-up of novel feedstock hydrolysis protocols producing a range of mono/oligosaccharides which are of significant commercial interest.
The project results demonstrated that the conversion of pretreated waste paper may be successfully converted to lactic acid, which is an important building block in Industrial Chemistry. Within the frame of Waste2Go this lactic acid has been further processed to n-butyl lactate which is widely used as a biodegradable solvent in agri-chemical and other applications. In addition, the novel sub/supercritical water oxidation technology was investigated and a custom-built 1 litre autoclave was designed and developed by Separex (FR) for further processing to fractionate and/or purify the waste cellulosic material resulting from the degradation process in order to obtain several cello-oligomeric fractions. Beyond the cutting-edge scientific results there are a number of serious economic impacts of the project. Waste2Go will improve the competitiveness of European SMEs (5 SMEs in the consortium) in the waste management sector, which has the highest proportion of SMEs and the lowest proportion of valued added of any EU industrial sector. Valorisation of waste encourages practices which create a circular economy and will ultimately reduce dependency on imported raw materials, as well as decouple process input costs from the price of fossil oil, which mitigates risks associated with feedstock availability, price fluctuation and supply bottlenecks.
Project Context and Objectives:
In order to provide the context for the Waste2Go, the first step was to establish the scale and nature of waste available to feed into the process. In order to determine the scale of the resource a literature study was conducted, to establish the current EU waste profile and what treatment/disposal technologies were currently being employed. The output from this study is available as a Pdf. from www.waste2go.eu/downloads.
The objectives of investigation were to establish, what the current situation was across the EU regarding:
1. Regulation of waste management systems (waste legislation)
2. Waste management systems (collection, treatment and disposal)
3. Prices for disposal of MSW
4. Composition and amount of MSW including biogenic content
5. Variation of the biogenic content of MSW between the different countries
6. Chemical and physical characteristics of MSW, respectively energy potential and cellulose content of the biogenic substance
The waste hierarchy has been use as a driving force for the waste management industry emphasising the avoidance and reduction of waste as the preferred method of waste management, with disposal being the least preferable option. Waste management systems, were found to vary widely throughout the EU, with varying levels of waste segregations leading to vastly differing approached to the subsequent collection, treatment and disposal of waste streams. This variety in the waste treatment also led to vastly different prices for the disposal of waste, related to:
Level of technology used
Existing restrictions or bans for final disposal into landfills
Aftercare and remediation of landfills
Flue gas treatment of incineration plants
Collection system respectively for sorting of co-mingled waste fractions
There was a notable difference in the disposal prices for wastes from the northern and western EU members which tend to employ a higher level of technologies in disposing of waste streams and the relatively cheap Eastern European states the majority of which had relatively cheap disposal costs (European Environment Agency (EEA): Managing municipal solid waste – a review of achievements in 32 European countries (EEA report, No 2/2013). Publications Office of the European Union, Luxembourg (2013).
One of the major issues with Municipal Solid Waste (MSW) is that it is an ill-defined heterogeneous substrate, which is incredibly inconsistent by its very nature as being comingled waste from municipalities. Some of the key variables in the streams are:
Calorific content
Moisture content
Cellulosic content
Inhibitors for microorganisms/enzymes
As all of the above parameters will vary considerably dependent on the source of the MSW as well as any geographical, seasonal and cultural influences producing a biological system which can deal with the totality of MSW is not viable. Once this is combined with the challenges associated with waste market which is generally a relatively low priority for the majority of the engineering and scientific community such as:
Waste collection market ‘fractured’
SME’s in the area operate in niche markets with tight margins so often willing but not able to support research
Facilities tend to be waste stream specific
Even ‘defined’ waste streams vary massively in composition
The Waste2Go process targets the biogenic fraction of MSW, so for unsorted wastes, this would require the removal of recyclable materials such as plastics, metals and glass for example as these materials are not accessible to the enzymatic systems. The use of Rotaclave technology meant that unsorted waste streams – ‘black bag waste’ can be broken open so that the biogenic fraction is partially hydrolysed to make it easier for the enzymes to access the structures, while also sterilising materials such as metals and glass which can then be segregated using existing sorting technologies such as magnets and trammels.
The duration of the treatment in the rotaclave affected the physical properties of the final material. Work was undertaken to investigate how the operational parameters of the rotaclave process affected the hydrolysability of the final material by enzymatic processes. Even for a ‘model’ substrate such as waste paper there was batch to batch variation observed again highlighting the challenges with using MSW as a feedstock. A number of process trails were undertaken to establish an optimal process which produced a readily hydrolysable material without excessive operating time or costs.
The key to sustainable processing and valorization of MSW is having access to appropriate enzymes. The next step is then to find the right combination of biomass pretreatment and subsequent enzymatic treatment, to obtain the products that one is after. In the case of Waste2Go, the desired products were oligomeric sugars derived from polymeric sugars in the biogenic fraction of MSW (primarily composed of cellulose and hemicellulose). For benchmarking MSW degradability, and for exploring alternative application routes, enzymatic conversion to monosugars was also explored. Obtaining the first goal required development of in-house enzyme cocktails, whereas the second goal was pursued using commercial enzyme preparations.
The Enzyme Platform was to develop on the organisms and systems developed as part of the enzyme trial work and then scale up their production to feed subsequent work packages.
Objectives
»» Develop fermentation and downstream recovery processes to supply cellulolytic enzymes of interest to WP3 and WP5.
»» Define and demonstrate production processes at 750 L scale
The economic viability of Industrial Biotechnology (IB) processes based on sustainable biomass feedstocks is, to a large extent, reliant on the enzymatic hydrolysis of diverse substrates (e.g. lignocellulosic biomass, paper pulp, forestry waste, cardboard, food waste) at high solids loadings (>20% w/v); which poses operational challenges. Efficient feedstock hydrolysis requires turbulent mixing which, in general, cannot be achieved with impellers in conventional stirred tank reactors, leading to sub-optimal process operations and economics. As a result a novel reaction vessel was developed in order to process high solids loaded slurries. This reactor enabled the mixing of the pre-treated MSW with enzymes at high solids loading's, while ensuring that there was sufficient mixing to promote enzyme activity.
The post enzyme treated slurries, solids and liquids were then taken and processed to separate and fractionate discreet product fractions which could be used to replace petrochemical derived materials. The super/sub critical water apparatus also proved to be able to hydrolyse untreated MSW streams.
The final work packages assessed the technologies employed against the Best Available Technologies (BAT) in waste treatment to assess the viability of the Waste2Go process as a disruptive technological solution to the issue of MSW.
Project Results:
At the start of the project an enzyme platform was built up, primarily by combining the expertise of NMBU and CPI. We selected candidate enzymes by careful studies of the literature and bioinformatic analyses, we established a Pichia-based expression platform, and produced enzymes in large scale for subsequent biomass processing. Developing the Pichia expression platform involved several inventive steps leading to a joint NMBU-CPI publication and to routine production of the desired enzymes. Notably, as part of establishing the enzyme platform, important new enzyme properties were discovered, with significant scientific impact. For example, in a 2014 paper in PNAS, we describe a novel activity for a so-called “lytic polysaccharide monooxygenase” (LPMO), a redox enzyme that catalyses the oxidative degradation of polysaccharides. Today, we know that this enzyme can be used for highly innovative engineering of glucoconjugates. The full exploration of this LPMO’s application could not be done within the framework of Waste2Go resources, but is currently considered highly promising for biomass valorization and is being pursued in other projects.
In the next phase of the project, in iterations between the WP2 (pretreatment), WP3 (enzyme platform) & WP4 (enzyme production), a large amount of effort was used to convert our model substrates, confidential waste paper (CWP) and dry, mixed recycling (DMR), to soluble cellooligomers (= oligomeric sugars derived from cellulose), or to other potentially useful compounds, other than glucose, such as cellobionic acid. This work was considered risky from the start of the project and turned out to be even more difficult than expected. Basically, after a large series of experiments, this approach was halted. At that time, some interesting options remained unexplored, but we considered it to be more important to embark on alternative strategies.
Two alternative strategies were explored to utilize the cellulosic fraction of MSW, the second of which, the second was anticipated in the original proposal and entails key technological developments that were anticipated as being a key result of Waste2Go.
Strategy 1,
Complete saccharification of the cellulose. This goal was pursued to verify the degradability of the pretreated materials provided by WP2. This was essential to monitor progress in WP2 and identify optimal pretreatment strategies. Furthermore, during the course of the project, it became clear that fully depolymerized material was of interest to one of the key industrial partners, Chemoxy. This work was very successful in that complete saccharification of input materials was indeed achieved at reasonable enzyme loadings. These important results ensured acceptable progress in WP5 (bioprocess development) and WP7 (Derivatisation).
Strategy 2,
Sequential dissolution-precipitation of cellulose using different physical and chemical principles. In this approach, we aimed at isolating fractions of longer, water-insoluble oligomeric cellulose with a discrete narrow molecular weight range. In a collaboration between NMBU, CPI and Feyecon, enzyme-treated MSW was sequentially dissolved in organic/inorganic solvents by varying a critical dissolution parameter in consecutive dissolution steps. We evaluated four established technologies for dissolution-precipitation of cellulose (described in the original proposal) for their potential to fractionate cellulose into a range of discrete narrow molecular weight fractions of oligomeric materials. This strategy allowed us to successfully isolate oligomeric cellulose fractions, some of which are processed further for the downstream production of surfactants (i.e. glycoconjugates).
In addition to cellulose, we also assessed the utilization of hemicellulose occurring in various biogenic (plant-based) fraction of MSW, targeting primarily the production of
hemicellulosic oligosaccharides. Firstly, we developed a method to produce glucuronoxylo-oligosaccharides from xylan (figure 3.6). Xylan comprises 20-30% of the dry weight of hardwood that can occur for example in yard trimmings. We successfully established a model system for pretreatment and enzymatic treatment of wood chips from hardwood species, using steam explosion and commercially available xylan-active enzymes. Based on our findings, we have drafted a manuscript that will be submitted shortly. Secondly, we targeted the production of an oligosaccharide from xyloglucan that occurs for example in vegetable waste. It is fully feasible to convert xyloglucan into a mixture of neutral xylogluco-oligosaccharides built up from 7-10 sugar monomers) and further into a uniform heptasaccharide, referred to as XG7. Such a heptameric oligomer can then be derivatized to a variety of glycoconjugates, including surfactants.
Producing green, biobased surfactants
A key idea of Waste2Go was to use the oligomeric carbohydrate products to produce environmentally friendly green surfactants, also known as biobased surfactants. One could achieve this by coupling the hydrophilic carbohydrate oligomer to a lipophilic compound, thereby generating a bi-functional molecule that has the abilities of lowering the surface tension of water and carrying hydrophobic compounds (hydrocarbons etc.) through aqueous environments. Since our goal of producing carbohydrate oligomers directly from MSW was not reached in time, a model system was employed as a proof-of-concept.
A xyloglucan-derived heptasaccharide (herafter XG7) made by a commercial producer was used as a model substrate to demonstrate the chemical reactions resulting in the synthesis of a biobased surfactant. In theory, any oligosaccharide generated in Waste2Go might undergo such a reaction scheme, if desired, and yield a wide variety of biobased surfactant products, where the chemical properties may be manipulated based on the size and confirmation of the oligosaccharide and the lipophilic group. Notably, an acidic xylo-oligosaccharide derived from woody MSW, would yield an even more attractive biobased surfactant, as it would, in addition to the hydrophilic part, also contain a negative charge. The negative charge would be capable of forming complexes with metals, for instance used in generating soaps and in remediation of soils polluted by heavy metals.
WP4 ran from January 2013 to December 2014, completing 137 fermentations. Enzymes selected as being of special interest needed in “enzyme cocktails” were cloned into Pichia pastoris yeast by NMBU. The processes for growing the yeast, producing the enzymes and recovering the enzymes were developed at small scale up to 10L. In the development stage, parts of the process were improved and challenges overcome before scaling up to 750L pilot plant scale.
Two LPMOs, three endoglucanases and a cellobiose dehydrogenase were produced at 750L scale and made available for the work on cellulosic degradation. The production processes were defined and are sufficiently flexible to accommodate future enzymes cloned into similar host strains.
Enzyme production at plant scale
P. pastor is fermentation and enzyme recovery processes
In the “GAP” system, which is a so-called “constitutive expression system”, Pichia cells constantly make enzyme as they grow, secreting them out of the cell. Production of the enzymes by a fermentation process starts with progressing a sequence of small seed cultures until the volume is sufficient to seed the main fermenter, which will contain a final volume of ~750L in a 1000L stainless steel vessel. Temperature and acidity are then monitored and regulated at the required setpoints. After 24h, glycerol, the carbon and energy source for the yeast, is fed in a controlled and ramped manner over 3 days to facilitate growth at such a rate that balances productivity with overgrowth which can cause foaming and over pressurisation.
As the cells multiply, they produced the desired enzyme which accumulated in the broth. The “AOX” system differs from the GAP system in that it is an “inducible expression system” – that is to say the enzyme desired is only produced when the system is switched on by the introduction of an inducer, in this case methanol. The fermentation process to make enzyme is similar to the GAP-based one with 24h of growth followed by ramped growth controlled by a glycerol feed. This time, however, the glycerol feed only lasts 24h rather than 3 days. For the last 2 days, when sufficient biomass has been generated, glycerol is replaced by methanol which acts
as both a substrate for yeast growth as well as switching on enzyme production. During this stage, enzyme accumulates in the broth.
Whichever fermentation process was employed, the downstream processing to recover the enzyme is the same Essentially, the cells are removed by centrifugation, and the liquid containing the enzyme is processed by cross-flow filtration to concentrate the enzyme and buffer it at the right pH. Finally, the enzyme stocks are frozen until needed for use in the cellulose degradation processes.
The economic viability of Industrial Biotechnology (IB) processes based on sustainable biomass feedstocks is, to a large extent, reliant on the enzymatic hydrolysis of diverse substrates (e.g. lignocellulosic biomass, paper pulp, forestry waste, cardboard, food waste) at high solids loadings (>20% w/v); which poses operational challenges. Efficient feedstock hydrolysis requires turbulent mixing which, in general, cannot be achieved with impellors in conventional stirred tank reactors, leading to sub-optimal process operations and economics.
The Biomixer developed at CPI, with a fill volume of 100 litres, overcomes many of these limitations through the application of a stainless steel horizontal drum containing a central shaft running along its length from which three radial paddles are attached. The paddles are powered by a 0.4 kW motor with an inline step down gearbox providing high torque even at low rotation speeds. This produces a lift and drop, tumbling action which ensures mixing to homogeneity for high percentage solids materials. Furthermore, the paddles, which can be easily interchanged to test new mixing strategies, have been designed for excellent performance in the presence of solids in the substrate but continue to efficiently mix the product after liquefaction has occurred. Temperature control was essential in order to achieve optimal reaction conditions for enzymatic feedstock hydrolysis. Conventionally, mixing of high solids slurries is achieved through rotation of the entire vessel (e.g. cement mixer) resulting in limited mechanisms for temperature adjustment, such as steam injection.
In the CPI design, a water jacket capable of ramping the contents’ temperature at 40°C hour-1 is employed and can maintain isothermal conditions up to 90°C. Despite its ability to operate at high temperatures, the Biomixer is a non pressurised vessel with a range of features which permit safe operation. These include emergency stops, an interlock which prevents operation unless the hinged door is securely closed and gearing that allows the entire vessel to be tilted forwards and backwards, allowing facile loading/ unloading, reducing risks involved with manual handling.
Crucially for process development and pilot work, a data recorder charts a range of Biomixer parameters including power consumption, pH, temperature and shaft rotation speed. This inline monitoring provides invaluable information for process optimisation in relation to substrate characteristics over time and overall energy efficiency. Moreover, the plug and play nature of the Biomixer design allows for new probes to be installed and their outputs recorded with a minimum of down time. The Biomixer was taken from concept to delivery in six months enabling the Waste2Go project to process a range of substrates at high solids loadings in excess of 20% dry weight (w/v) which would not have been feasible in conventional stirred tank reactors. As such it has expedited the scale up of novel feedstock hydrolysis protocols utilising a range of feedstock types to produce both soluble and insoluble cellulosic sugars of commercial interest. These fractions have been provided to the Waste2Go project partners who have built upon the outputs from WP5.
Purification and separation
The depletion of the mineral oil resources together with the environmental problems that the use of fossil fuels entails makes the search for alternative sources that can be used for the production of chemicals and fuels imperative. In this sense the use of biomass represents an excellent alternative due to its renewable nature and wide availability. The strong dependence on fossil fuels for the production of both energy and chemicals is one of the main environmental and political concerns worldwide. Even though some several renewable energy technologies are currently under development or already available, such as solar or wind energy, for the production of fuels and chemicals biomass seems to be the only feasible renewable source due to its high carbon content. Biomass is formed by cellulose, hemicellulose and lignin, biopolymers that have a great potential for the production of valuable chemicals.
Those plants, however, which are most easily converted into fuel and chemicals, are food plants. In order to avoid using food as fuel, the focus has shifted to the biogenic
part contained in the municipal solid waste (MSW). This also imparts other advantages such as the valorization of the waste, and reducing its socio-economic cost and environmental impact. Valorization of the biomass contained in the MSW comprises several steps that include both thermo-mechanical and biodegradation processes. However, the product resulting from the biodegradation process is characterized by a wide range of molecular weights. Therefore, this material needs to be fractionated and purified in order to obtain several fractions of narrower molecular weight ranges that can be used as feed-stocks in the industry or as starting materials for the production of other chemicals such as surfactants. FeyeCon Group (FeyeCon D&I and Separex SAS), within the Waste2go project, has investigated and developed four different technologies in order to fractionate and purify the cellulosic material obtained after the biodegradation treatment of MSW. All these technologies were first prototyped and tested by using α-cellulose as substrate
in order to set-up the methods. Once their suitability for cellulose fractionation was proven, the same technologies were used to treat the MSW material and produce different fractions with different potential.
Three of these technologies are based on solvent solutions: metal complexes (LiCl/DMAc), ionic liquids, and aqueous alkali metal hydroxide solutions. These systems are known to dissolve cellulose, which is sparingly soluble in most common solvents. However, just dissolving the cellulose is not enough. In addition it is necessary to develop the process in such a way that different fractions with different molecular weight ranges can be obtained by varying certain key parameters. Mixtures of lithium chloride in dimethymacteamide (LiCl/DMAc) have been extensively reported in the literature as suitable solvents for cellulose with the concentration of LiCl being the limiting parameter to control cellulose dissolution at a given temperature. We were able to show that higher concentrations of LiCl, the longer cellulosic chains. Due to this, it is possible to obtain different fractions of different average degrees of polymerization (DP) by consecutive dissolution of the cellulosic material in LiCl/DMAc solutions containing increasing amounts of the salt.
Aqueous sodium hydroxide solutions are also suitable solvents for cellulose when the concentration of NaOH lies within a certain range. The other key parameter that
influences the polymer solubility in these solvent mixtures is the temperature: low temperatures are required for dissolution since at room temperature cellulose does not dissolve but swells. By varying the dissolution temperature it was possible to obtain several fractions of different DP from the same raw material. Pure aqueous NaOH was found to be a poor solvent for cellulosic materials; however, additives such as urea and thiourea enhance the solubility leading to higher yields. There is a wide variety of ionic liquids that can dissolve cellulose successfully. However, dissolution of the polymer in these solvents will just lead to a negligible decrease in the DP of the polymer. In order to depolymerize the cellulose an acidic catalyst needs to be added to the mixture. The catalyst and ionic liquid employed in this technology were selected jointly taking into account several limiting factors such as the viscosity and melting point of the ionic liquid, solubility of the cellulose in the ionic liquid and stability of the catalyst in the solvent. On the other hand the catalyst should be heterogeneous, so as to avoid tedious separation processes, and should possess a macro porous structure with a large surface area in order to overcome the mass transfer limitations arising from the high viscosity of the mixture. Fractions containing different average degree of polymerization could be obtained by varying the reaction time. The longer the polymer is in contact with the catalyst the further depolymerization proceeds and, therefore, the lower the molecular weight present in the sample obtained.
The forth technology that has been developed for cellulose fractionation is super- or subcritical water. Supercritical water, that is water at a temperature and pressure above its critical point (374.1°C and 22.1MPa) represents a promising alternative for cellulose valorization due to its excellent solvation properties for organic compounds as well as the fact that it can act itself as an acid catalyst thus depolymerizing he cellulose without the need of another catalyst. However, the reaction is difficult to control. It is extremely fast, and often results in the total degradation of the substrate. Also, the fractionation in supercritical water requires specific equipment that has been designed and built specifically for this project by Separex SAS.
Fractionation of α-cellulose was carried out at supercritical conditions leading to the total degradation of the polymer at already one minute reaction time. Due to this it was decided to use subcritical conditions instead. Subcritical water, liquid water at temperatures between the atmospheric boiling point and the critical temperature, shares many characteristics that make subcritical water a suitable for cellulose depolymerization (it is a good solvent for cellulose and can act as an acid catalyst), however, the reaction speed is lower in this case, which makes the reaction easier to control.
Different types of feeds were treated at subcritical conditions: α-cellulose, TM-MSW
(thermo-mechanically treated but non-biodegraded material), and TM–MDSW-D (thermo-mechanically and enzymatically degraded material). In the three cases the treatment with water under subcritical conditions led to the decrease of the average DP of the materials and the production of cellodextrin mixtures. This technique is very innovative and brings two important advantages for the valorization of the municipal solid waste: it does not require a costly pre-drying step and it can lead to the destruction of non-desirable compounds that might be present in the waste material.
Potential Impact:
Life cycle assessment (lca) is an ISO 14040/44 standardised method to assess the environmental impact of products, processes and services along their value chain including the production, the use and the end of life phase. The lca method and the gained results support decision makers like politicians, individuals, and engineers to include environmental aspects during different life cycle phases and to identify and then minimize potential shift of burdens.
To determine the environmental impact of first, the individual processes of the Waste2Go approach and second, the Waste2Go approach itself, a systematic collection of all mass and energy flows has been done. The flows were confirmed and quantified by the project partners, transferred into the GaBi software and the environmental impact calculated. Subsequent the process with the highest significant environmental impact was determined. By means of this information engineers could
include environmental aspects early in the development phase to redesign ecological drivers to minimize the environmental impact.
The enzyme production process was calculated to have the highest environmental impact of all of the treatment steps employed accounting for roughly 70% of the total environmental impact of the process across a number of measurements. Based on the actual state of the technology development upscaling scenarios were performed for the enzyme production, the biodegradation process and the subcritical treatment.
Based on the upscaling assumptions it would possible to reduce a huge amount of CO2-equivalents emitted by the Waste2Go approach. It is worth to concentrate on the reduction of the energy demand and to reuse the enzymes as they are the main drivers. Especially the reusing of the enzymes shows a high potential to reduce environmental impacts. This is explained by the significance of the enzyme production process.
Dissemination and exploitation
Work was undertaken to facilitate the impact of the research outputs in its commercial exploitation by duly protecting intellectual property. The partners joined this project to develop the technology and to commercially benefit from its wider uptake and commercialisation so were therefore actively involved in dissemination activities. Whilst due to budgetary constraints the allocation of person-months per participant in the request for funding from the EU had been restricted, the partners all contributed additional resources at their own expense to ensure maximum impact.
As part of the dissemination activities it was important to ensure that the protocols developed included ongoing protection of potential foreground IP, to plan for post project exploitation and to undertake dissemination actions that enable the partners to maximise the commercialisation of the project results. The following activities were planned for the project lifetime:
• Three international seminars with a total audience in excess of one hundred and fifty attendees
• The submission of more than ten articles for publication
• More than two hundred and fifty visits to the public facing pages of the project web site and
Dissemination and communication activities in Waste2Go commenced from the very beginning of the project to raise awareness of the target groups on the objectives and the upcoming activities of the project from which they could benefit, as well as to engage them in those activities. A visual identity for Waste2Go was developed right after the project commenced and related graphical elements were integrated into the specific dissemination and communication tools.:
The Dissemination Protocols for the project were developed at the early stage of the project as an intra-consortium document, has served as guidance for the Waste2Go partners to conduct more effectively the dissemination and communication activities.
Various means and tools of dissemination and communication have been developed as part of the Dissemination and Exploitation work package in Waste2Go. The following specific means and tools have been conceived and utilised by the partners:
• Project homepage (Waste2Go website) – www.waste2go.eu
• Project brochure
• Project poster
The Waste2Go Website
The Waste2Go website (www.Waste2Go.eu) was launched in January 2013, shortly after the project’s kick-off meeting and has been online for over 38 months. It provides information about the objectives, activities and expected results of the project, presents the project partners, and includes News and Downloads areas that had been duly updated to facilitate the visitors with the latest news and publicly available documents and deliverables generated by the project activities.
Web statistics help to better understand user behaviour and fields of interest when visiting the website, thus facilitating more focussed dissemination and content development. The following graphs indicate some basic statistics regarding the hits on the project website since its launch and concluding at closure.
Total number of visits at Waste2Go’s homepage since the launch of the site in April 2013 reached beyond 5800+ of which around 4100+ are unique visitors (exceeding by far the initially planned visits to the site). The value of unique visitors refers to the number of different IP addresses which were used to visit the Waste2Go web site, while the number of visits describes the total number of visits which were carried out in the given period including multiple visits from the same IP address. The pages value explains the total number of opened sub-sites within waste2go.eu while the bandwidth refers to the total amount of data which was downloaded from the site, including the available deliverables, brochures, etc.
The project’s outreach wasn't limited to the participating countries, but also attracted some interest towards its activities at other EU countries as well as at countries beyond the EU, most likely due to the fact that some high profile international conferences were attended by the representatives of the consortium and their contribution initiated interest towards the Waste2Go activities.
Waste2Go brochure
A project brochure (electronic and hardcopy) was produced right after the project started and was distributed at relevant events that were attended during the project duration by the partners and is available at the public section of the project website. It contains the overall aim of the project and the actions that are undertaken to achieve this aim, as well as the results from the completed WPs and the expected outcomes from WPs. It also contains a link to the homepage of Waste2Go, the general inquiry e-mail address as well as the contact details of the partners.
Waste2Go Poster
There were two different versions of the poster created for two distinct purposes one to provide a more general overview of the project’s activities and the other being more specific about the Life Cycle Assessment activities (related to the project’s sustainability work package). Following this approach there were a number of other poster versions generated primarily by Geonardo to suit the different needs of those partners who were participating at international or local events. These posters helped raise awareness of the project and its activities at a number of high profile events such as at the “Research & Innovation for a circular economy in European Regions”.event in Brussles or the New Zealand Life Cycle Assessment Conference 2014 in Wellington, New Zealand, just to name a few. (full the list of dissemination activities please refer to the summary table.
Waste2Go Press releases
The Consortium aimed to inform the wider public about major achievements of the project that were not subject of IP protection . For this reason, 3 set of press releases were drafted and circulated online to raise awareness on the projects activities and to initiate potential future collaborations for the project partners with non consortium enterprises. The first press release was covering the launch of the project and its website and the second focused on the both new hardware and the biomixer that had been developed at CPI within the scope of WP5 activities. The last press release focused on spreading the word about the final industry seminar held in Brussels in September 14, 2015.
Waste2Go on the web
Waste2Go partners were active in disseminating and promoting the project as a whole and their project-related activities on various online media channels.
Waste2Go Articles for Publication
By the end of the project, the following scientific papers were submitted by the partners on the project findings. Please note that some of these manuscripts will have been published after M36 but they were either approved by the journal or at least they had been submitted for review before the project ended.
• Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase. Biely P, Cziszárová M, Uhliariková I, Agger JW, Li XL, Eijsink VG, Westereng B. Biochim Biophys Acta. 2013 Jul 24; 1830(11):5075-5086.
• Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose. Peter Biely , Mária Cziszárová , Jane W. Agger , Xin-Liang Li , Vladimír Puchart , Mária Vršanská , Vincent G.H. Eijsink , Bjorge Westereng. Biochim Biophys Acta. 2014 Jan;1840(1):516-25.
• Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. J. W. Agger , T. Isaksen , A. Varnai , S. Vidal-Melgosa , W. G. T. Willats , R. Ludwig , S. J. Horn , V. G. H. Eijsink , B. Westereng. Proc Natl Acad Sci U S A. 2014 Apr 29;111(17):6287-92.
• Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Anikó Várnai , Campbell Tang , Oskar Bengtsson , Andrew Atterton , Geir Mathiesen , Vincent GH Eijsink. Microb Cell Fact. 2014 Apr 18;13(1):57.
• A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Stjepan Krešimir Kračun , Julia Schückel , Bjørge Westereng , Lisbeth Garbrecht Thygesen , Rune Nygaard Monrad , Vincent G H Eijsink, William George Tycho Willats. Biotechnol Biofuels. 2015 Apr 23;8:70.
• Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite -1. Tao Feng, Kok-Phen Yan, Maria D. Mikkelsen, Anne S. Meyer , Henk A. Schols, Bjørge Westereng, Jørn D. Mikkelsen. Appl Microbiol Biotechnol. 2014 Dec;98(23):9667-79
• Recent Progress in Understanding the Mode of Action of Acetylxylan Esterases. – NMBU - Article citation: J. Appl. Glycosci., 61, 35-44 (2014).
• The effect of paper source and pretreatment conditions on the enzymatic degradability of waste paper to mono- and oligosaccharides. – NMBU – Manuscript in preparation
• Biosurfactant production from a xyloglucan-derived oligosaccharide. – NMBU – Manuscript in preparation
• Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. – NMBU – submitted for publication
• Comparison of the roles of endoglucanases and LPMOs in the liquefaction of waste paper. – NMBU – Final data acquisition is on-going to finalise and submit the manuscript.
• Comparison of four methods to fractionate cellulose based on degree of polymerization. – NMBU – Final data acquisition is on-going to finalise and submit the manuscript.
Dissemination to the wider public
Following internal publication of the results, protection of all Foreground IP and the publication of scientific papers, the partners aimed to disseminate the Waste2Go project results to the wider public through the internet, TV, newspapers, radio, the popular scientific press. Each partner was be responsible for identifying opportunities for dissemination at a local, regional or national level and advising Geonardo, the WP leader, of these opportunities which would then be reviewed for implementation by the PSC.
Despite the highly technical nature of the core of research within the Waste2Go consortium, with special attention NMBU were able to deliver the message to the wider public in a fashion that was comprehensive for the non-specialist target group as well. The staff carried out a number of activities targeting the non-specialist public to ensure that the goals of the project reached beyond the ears of the scientific community. For this reason the NMBU crew gave a number of oral presentations targeting the policy makers and the general public and published articles on national level press:
• 03.04.2014 Vincent Eijsink; Enzymteknologi for norsk bioraffinering (NorZymeD); invitert foredrag på kontaktseminar mellom TINE og forskningsmiljøer; Olso, Norge. (Presentations in Norwegian for the Norwegian dairy industry; appr. 30 attendants)
• 04.04.2014 Vincent Eijsink; Mulige og aktuelle områder for FoU for realisering av skogens potensialer; foredrag på et lokalt møte om SKOG22 prosessen. Ås, Norge. (Presentation in Norwegian on the valorisation of forest resources)
• 22.05.2014 Vincent Eijsink, Nye enzymer for å utnytte det grønne karbonet; invited lecture in Norwegian at the 2014 «Energikonferansen», Oslo, Norway. Approximately 200 participants from research institutions, companies and governmental organizations, including high-level politicians.
• 26.05.2014 Vincent Eijsink, Using enzymes for valorizing biomass; an overview of the NorZymeD project and enzyme applications in biorefining. Invited lecture at the annual seminar of the Cycle project, Hell, Norway. Appr. 40 participants from research institutions, companies and governmental waste processing organizations.
• 30.10.2014 Vincent Eijsink, «Enzymteknologi i bioraffinering – Muligheter for fremtidig verdiskapning»; Presentasjon for lederen i SKOG22, Ås, Norway, Oct 30, 2014. (Presentation in Norwegian for the leader of a Governmental group working of the valorization of forest resources).
• 28.04.2014; I fremtiden spiser vi trær; NMBU webpages (Bjørge Westereng).
• 05.05.2014; I framtiden kan vi ha trær på menyen; article in Nasjonen (national newspaper) (Bjørge Westereng).
• 06.05.2014: I fremtiden spiser vi trær; article in Dagsavisen (national newspaper) (Bjørge Westereng).
Waste2Go also released a one page project introduction in the Parliament Magazine’s GreenWEEK edition in June 2015. The Parliament Magazine is the policy magazine of the European Union acting also as the Media for the whole of GreenWeek 2015, Europe’s biggest and most important environmental policy conference. The Magazine was distributed within the European parliament thus reaching all MEPs and political groups, EU council presidency officials, Council of ministers, senior officials; European commission, all EU commissioners and chefs de cabinet; Economic and Social Committee, some members in Brussels; Committee of the Regions, Brussels secretariat; European Court of Justice, senior officials; European Investment Bank, senior officials . Over 50,000 copies were distributed in total.
The link to the Waste2Go article can be located here:
https://www.theparliamentmagazine.eu/articles/magazines/issue-413-1-june-2015
The overall effectiveness and impact of the dissemination and communication activities in Waste2Go was considered to be both of a high standard and effective for the three-year duration of the project. Partners’ activities appropriately covered regional dissemination potential in terms of conferences participation, project promotion to the target groups and liaison with other relevant stakeholders. For more details, please refer to Annex E of this report.
The project’s international exposure reached the high standards set prior to the launch of the initiative. The international conferences significantly contributed to the website visits from non-European countries. In line with mid-term expectations, the national and international coverage of the project became more frequent as a result of both increased project activity and the delivery of cutting edge scientific results.
The partners were active throughout the project, attending national, regional, European even oversea events to promote Waste2Go and its activities to the relevant target groups. The second half of the project saw a more targeted dissemination and communication by all partners. The table In Annex E provides an exhaustive list of list events that Waste2Go partners attended over the whole duration of the project.
List of Websites:
http://www.waste2go.eu/
Each year, over 180 million tonnes of Municipal Solid Waste (MSW) are discarded in the EU, which is in excess of 1kg per citizen every day. Technologies such as incineration, anaerobic digestion and composting all have a role to play in the processing of this waste but these technologies fail to exploit the chemical value contained in this material. This is where Waste2Go comes in.
Waste2Go (www.waste2go.eu) is an EU funded research and development project that is developing technologies to improve waste management and increase value –social, economic and environmental. Its main objective is the innovative transformation of the biogenic fraction of MSW, which accounts for over 55%, into chemical with an economic value greater than its use as an energy source. During the 3-year project the primary focus was on transforming specific organic waste streams such as waste paper, dry mixed recycling and black bin bag waste to useful chemical feedstocks. Once various strains of microorganisms were developed at the Norwegian University of Life Sciences for producing cellulose degrading enzymes research was shifted to the UK, where CPI developed small-scale industrially-relevant processes for cell growth and enzyme production. With knowledge gained from this work, the production processes were then successfully run at 750L scale, yielding sufficient stocks of six key enzymes to enable the project to use them in trials for the conversion of biogenic waste cellulose to valuable chemicals. In parallel a bespoke mixing vessel was also developed which enabled the project to process a range of waste derived substrates at high solids loadings in excess of 20% dry weight (w/v) which would not have been feasible in conventional stirred tank reactors. This engineering feat has expedited the scale-up of novel feedstock hydrolysis protocols producing a range of mono/oligosaccharides which are of significant commercial interest.
The project results demonstrated that the conversion of pretreated waste paper may be successfully converted to lactic acid, which is an important building block in Industrial Chemistry. Within the frame of Waste2Go this lactic acid has been further processed to n-butyl lactate which is widely used as a biodegradable solvent in agri-chemical and other applications. In addition, the novel sub/supercritical water oxidation technology was investigated and a custom-built 1 litre autoclave was designed and developed by Separex (FR) for further processing to fractionate and/or purify the waste cellulosic material resulting from the degradation process in order to obtain several cello-oligomeric fractions. Beyond the cutting-edge scientific results there are a number of serious economic impacts of the project. Waste2Go will improve the competitiveness of European SMEs (5 SMEs in the consortium) in the waste management sector, which has the highest proportion of SMEs and the lowest proportion of valued added of any EU industrial sector. Valorisation of waste encourages practices which create a circular economy and will ultimately reduce dependency on imported raw materials, as well as decouple process input costs from the price of fossil oil, which mitigates risks associated with feedstock availability, price fluctuation and supply bottlenecks.
Project Context and Objectives:
In order to provide the context for the Waste2Go, the first step was to establish the scale and nature of waste available to feed into the process. In order to determine the scale of the resource a literature study was conducted, to establish the current EU waste profile and what treatment/disposal technologies were currently being employed. The output from this study is available as a Pdf. from www.waste2go.eu/downloads.
The objectives of investigation were to establish, what the current situation was across the EU regarding:
1. Regulation of waste management systems (waste legislation)
2. Waste management systems (collection, treatment and disposal)
3. Prices for disposal of MSW
4. Composition and amount of MSW including biogenic content
5. Variation of the biogenic content of MSW between the different countries
6. Chemical and physical characteristics of MSW, respectively energy potential and cellulose content of the biogenic substance
The waste hierarchy has been use as a driving force for the waste management industry emphasising the avoidance and reduction of waste as the preferred method of waste management, with disposal being the least preferable option. Waste management systems, were found to vary widely throughout the EU, with varying levels of waste segregations leading to vastly differing approached to the subsequent collection, treatment and disposal of waste streams. This variety in the waste treatment also led to vastly different prices for the disposal of waste, related to:
Level of technology used
Existing restrictions or bans for final disposal into landfills
Aftercare and remediation of landfills
Flue gas treatment of incineration plants
Collection system respectively for sorting of co-mingled waste fractions
There was a notable difference in the disposal prices for wastes from the northern and western EU members which tend to employ a higher level of technologies in disposing of waste streams and the relatively cheap Eastern European states the majority of which had relatively cheap disposal costs (European Environment Agency (EEA): Managing municipal solid waste – a review of achievements in 32 European countries (EEA report, No 2/2013). Publications Office of the European Union, Luxembourg (2013).
One of the major issues with Municipal Solid Waste (MSW) is that it is an ill-defined heterogeneous substrate, which is incredibly inconsistent by its very nature as being comingled waste from municipalities. Some of the key variables in the streams are:
Calorific content
Moisture content
Cellulosic content
Inhibitors for microorganisms/enzymes
As all of the above parameters will vary considerably dependent on the source of the MSW as well as any geographical, seasonal and cultural influences producing a biological system which can deal with the totality of MSW is not viable. Once this is combined with the challenges associated with waste market which is generally a relatively low priority for the majority of the engineering and scientific community such as:
Waste collection market ‘fractured’
SME’s in the area operate in niche markets with tight margins so often willing but not able to support research
Facilities tend to be waste stream specific
Even ‘defined’ waste streams vary massively in composition
The Waste2Go process targets the biogenic fraction of MSW, so for unsorted wastes, this would require the removal of recyclable materials such as plastics, metals and glass for example as these materials are not accessible to the enzymatic systems. The use of Rotaclave technology meant that unsorted waste streams – ‘black bag waste’ can be broken open so that the biogenic fraction is partially hydrolysed to make it easier for the enzymes to access the structures, while also sterilising materials such as metals and glass which can then be segregated using existing sorting technologies such as magnets and trammels.
The duration of the treatment in the rotaclave affected the physical properties of the final material. Work was undertaken to investigate how the operational parameters of the rotaclave process affected the hydrolysability of the final material by enzymatic processes. Even for a ‘model’ substrate such as waste paper there was batch to batch variation observed again highlighting the challenges with using MSW as a feedstock. A number of process trails were undertaken to establish an optimal process which produced a readily hydrolysable material without excessive operating time or costs.
The key to sustainable processing and valorization of MSW is having access to appropriate enzymes. The next step is then to find the right combination of biomass pretreatment and subsequent enzymatic treatment, to obtain the products that one is after. In the case of Waste2Go, the desired products were oligomeric sugars derived from polymeric sugars in the biogenic fraction of MSW (primarily composed of cellulose and hemicellulose). For benchmarking MSW degradability, and for exploring alternative application routes, enzymatic conversion to monosugars was also explored. Obtaining the first goal required development of in-house enzyme cocktails, whereas the second goal was pursued using commercial enzyme preparations.
The Enzyme Platform was to develop on the organisms and systems developed as part of the enzyme trial work and then scale up their production to feed subsequent work packages.
Objectives
»» Develop fermentation and downstream recovery processes to supply cellulolytic enzymes of interest to WP3 and WP5.
»» Define and demonstrate production processes at 750 L scale
The economic viability of Industrial Biotechnology (IB) processes based on sustainable biomass feedstocks is, to a large extent, reliant on the enzymatic hydrolysis of diverse substrates (e.g. lignocellulosic biomass, paper pulp, forestry waste, cardboard, food waste) at high solids loadings (>20% w/v); which poses operational challenges. Efficient feedstock hydrolysis requires turbulent mixing which, in general, cannot be achieved with impellers in conventional stirred tank reactors, leading to sub-optimal process operations and economics. As a result a novel reaction vessel was developed in order to process high solids loaded slurries. This reactor enabled the mixing of the pre-treated MSW with enzymes at high solids loading's, while ensuring that there was sufficient mixing to promote enzyme activity.
The post enzyme treated slurries, solids and liquids were then taken and processed to separate and fractionate discreet product fractions which could be used to replace petrochemical derived materials. The super/sub critical water apparatus also proved to be able to hydrolyse untreated MSW streams.
The final work packages assessed the technologies employed against the Best Available Technologies (BAT) in waste treatment to assess the viability of the Waste2Go process as a disruptive technological solution to the issue of MSW.
Project Results:
At the start of the project an enzyme platform was built up, primarily by combining the expertise of NMBU and CPI. We selected candidate enzymes by careful studies of the literature and bioinformatic analyses, we established a Pichia-based expression platform, and produced enzymes in large scale for subsequent biomass processing. Developing the Pichia expression platform involved several inventive steps leading to a joint NMBU-CPI publication and to routine production of the desired enzymes. Notably, as part of establishing the enzyme platform, important new enzyme properties were discovered, with significant scientific impact. For example, in a 2014 paper in PNAS, we describe a novel activity for a so-called “lytic polysaccharide monooxygenase” (LPMO), a redox enzyme that catalyses the oxidative degradation of polysaccharides. Today, we know that this enzyme can be used for highly innovative engineering of glucoconjugates. The full exploration of this LPMO’s application could not be done within the framework of Waste2Go resources, but is currently considered highly promising for biomass valorization and is being pursued in other projects.
In the next phase of the project, in iterations between the WP2 (pretreatment), WP3 (enzyme platform) & WP4 (enzyme production), a large amount of effort was used to convert our model substrates, confidential waste paper (CWP) and dry, mixed recycling (DMR), to soluble cellooligomers (= oligomeric sugars derived from cellulose), or to other potentially useful compounds, other than glucose, such as cellobionic acid. This work was considered risky from the start of the project and turned out to be even more difficult than expected. Basically, after a large series of experiments, this approach was halted. At that time, some interesting options remained unexplored, but we considered it to be more important to embark on alternative strategies.
Two alternative strategies were explored to utilize the cellulosic fraction of MSW, the second of which, the second was anticipated in the original proposal and entails key technological developments that were anticipated as being a key result of Waste2Go.
Strategy 1,
Complete saccharification of the cellulose. This goal was pursued to verify the degradability of the pretreated materials provided by WP2. This was essential to monitor progress in WP2 and identify optimal pretreatment strategies. Furthermore, during the course of the project, it became clear that fully depolymerized material was of interest to one of the key industrial partners, Chemoxy. This work was very successful in that complete saccharification of input materials was indeed achieved at reasonable enzyme loadings. These important results ensured acceptable progress in WP5 (bioprocess development) and WP7 (Derivatisation).
Strategy 2,
Sequential dissolution-precipitation of cellulose using different physical and chemical principles. In this approach, we aimed at isolating fractions of longer, water-insoluble oligomeric cellulose with a discrete narrow molecular weight range. In a collaboration between NMBU, CPI and Feyecon, enzyme-treated MSW was sequentially dissolved in organic/inorganic solvents by varying a critical dissolution parameter in consecutive dissolution steps. We evaluated four established technologies for dissolution-precipitation of cellulose (described in the original proposal) for their potential to fractionate cellulose into a range of discrete narrow molecular weight fractions of oligomeric materials. This strategy allowed us to successfully isolate oligomeric cellulose fractions, some of which are processed further for the downstream production of surfactants (i.e. glycoconjugates).
In addition to cellulose, we also assessed the utilization of hemicellulose occurring in various biogenic (plant-based) fraction of MSW, targeting primarily the production of
hemicellulosic oligosaccharides. Firstly, we developed a method to produce glucuronoxylo-oligosaccharides from xylan (figure 3.6). Xylan comprises 20-30% of the dry weight of hardwood that can occur for example in yard trimmings. We successfully established a model system for pretreatment and enzymatic treatment of wood chips from hardwood species, using steam explosion and commercially available xylan-active enzymes. Based on our findings, we have drafted a manuscript that will be submitted shortly. Secondly, we targeted the production of an oligosaccharide from xyloglucan that occurs for example in vegetable waste. It is fully feasible to convert xyloglucan into a mixture of neutral xylogluco-oligosaccharides built up from 7-10 sugar monomers) and further into a uniform heptasaccharide, referred to as XG7. Such a heptameric oligomer can then be derivatized to a variety of glycoconjugates, including surfactants.
Producing green, biobased surfactants
A key idea of Waste2Go was to use the oligomeric carbohydrate products to produce environmentally friendly green surfactants, also known as biobased surfactants. One could achieve this by coupling the hydrophilic carbohydrate oligomer to a lipophilic compound, thereby generating a bi-functional molecule that has the abilities of lowering the surface tension of water and carrying hydrophobic compounds (hydrocarbons etc.) through aqueous environments. Since our goal of producing carbohydrate oligomers directly from MSW was not reached in time, a model system was employed as a proof-of-concept.
A xyloglucan-derived heptasaccharide (herafter XG7) made by a commercial producer was used as a model substrate to demonstrate the chemical reactions resulting in the synthesis of a biobased surfactant. In theory, any oligosaccharide generated in Waste2Go might undergo such a reaction scheme, if desired, and yield a wide variety of biobased surfactant products, where the chemical properties may be manipulated based on the size and confirmation of the oligosaccharide and the lipophilic group. Notably, an acidic xylo-oligosaccharide derived from woody MSW, would yield an even more attractive biobased surfactant, as it would, in addition to the hydrophilic part, also contain a negative charge. The negative charge would be capable of forming complexes with metals, for instance used in generating soaps and in remediation of soils polluted by heavy metals.
WP4 ran from January 2013 to December 2014, completing 137 fermentations. Enzymes selected as being of special interest needed in “enzyme cocktails” were cloned into Pichia pastoris yeast by NMBU. The processes for growing the yeast, producing the enzymes and recovering the enzymes were developed at small scale up to 10L. In the development stage, parts of the process were improved and challenges overcome before scaling up to 750L pilot plant scale.
Two LPMOs, three endoglucanases and a cellobiose dehydrogenase were produced at 750L scale and made available for the work on cellulosic degradation. The production processes were defined and are sufficiently flexible to accommodate future enzymes cloned into similar host strains.
Enzyme production at plant scale
P. pastor is fermentation and enzyme recovery processes
In the “GAP” system, which is a so-called “constitutive expression system”, Pichia cells constantly make enzyme as they grow, secreting them out of the cell. Production of the enzymes by a fermentation process starts with progressing a sequence of small seed cultures until the volume is sufficient to seed the main fermenter, which will contain a final volume of ~750L in a 1000L stainless steel vessel. Temperature and acidity are then monitored and regulated at the required setpoints. After 24h, glycerol, the carbon and energy source for the yeast, is fed in a controlled and ramped manner over 3 days to facilitate growth at such a rate that balances productivity with overgrowth which can cause foaming and over pressurisation.
As the cells multiply, they produced the desired enzyme which accumulated in the broth. The “AOX” system differs from the GAP system in that it is an “inducible expression system” – that is to say the enzyme desired is only produced when the system is switched on by the introduction of an inducer, in this case methanol. The fermentation process to make enzyme is similar to the GAP-based one with 24h of growth followed by ramped growth controlled by a glycerol feed. This time, however, the glycerol feed only lasts 24h rather than 3 days. For the last 2 days, when sufficient biomass has been generated, glycerol is replaced by methanol which acts
as both a substrate for yeast growth as well as switching on enzyme production. During this stage, enzyme accumulates in the broth.
Whichever fermentation process was employed, the downstream processing to recover the enzyme is the same Essentially, the cells are removed by centrifugation, and the liquid containing the enzyme is processed by cross-flow filtration to concentrate the enzyme and buffer it at the right pH. Finally, the enzyme stocks are frozen until needed for use in the cellulose degradation processes.
The economic viability of Industrial Biotechnology (IB) processes based on sustainable biomass feedstocks is, to a large extent, reliant on the enzymatic hydrolysis of diverse substrates (e.g. lignocellulosic biomass, paper pulp, forestry waste, cardboard, food waste) at high solids loadings (>20% w/v); which poses operational challenges. Efficient feedstock hydrolysis requires turbulent mixing which, in general, cannot be achieved with impellors in conventional stirred tank reactors, leading to sub-optimal process operations and economics.
The Biomixer developed at CPI, with a fill volume of 100 litres, overcomes many of these limitations through the application of a stainless steel horizontal drum containing a central shaft running along its length from which three radial paddles are attached. The paddles are powered by a 0.4 kW motor with an inline step down gearbox providing high torque even at low rotation speeds. This produces a lift and drop, tumbling action which ensures mixing to homogeneity for high percentage solids materials. Furthermore, the paddles, which can be easily interchanged to test new mixing strategies, have been designed for excellent performance in the presence of solids in the substrate but continue to efficiently mix the product after liquefaction has occurred. Temperature control was essential in order to achieve optimal reaction conditions for enzymatic feedstock hydrolysis. Conventionally, mixing of high solids slurries is achieved through rotation of the entire vessel (e.g. cement mixer) resulting in limited mechanisms for temperature adjustment, such as steam injection.
In the CPI design, a water jacket capable of ramping the contents’ temperature at 40°C hour-1 is employed and can maintain isothermal conditions up to 90°C. Despite its ability to operate at high temperatures, the Biomixer is a non pressurised vessel with a range of features which permit safe operation. These include emergency stops, an interlock which prevents operation unless the hinged door is securely closed and gearing that allows the entire vessel to be tilted forwards and backwards, allowing facile loading/ unloading, reducing risks involved with manual handling.
Crucially for process development and pilot work, a data recorder charts a range of Biomixer parameters including power consumption, pH, temperature and shaft rotation speed. This inline monitoring provides invaluable information for process optimisation in relation to substrate characteristics over time and overall energy efficiency. Moreover, the plug and play nature of the Biomixer design allows for new probes to be installed and their outputs recorded with a minimum of down time. The Biomixer was taken from concept to delivery in six months enabling the Waste2Go project to process a range of substrates at high solids loadings in excess of 20% dry weight (w/v) which would not have been feasible in conventional stirred tank reactors. As such it has expedited the scale up of novel feedstock hydrolysis protocols utilising a range of feedstock types to produce both soluble and insoluble cellulosic sugars of commercial interest. These fractions have been provided to the Waste2Go project partners who have built upon the outputs from WP5.
Purification and separation
The depletion of the mineral oil resources together with the environmental problems that the use of fossil fuels entails makes the search for alternative sources that can be used for the production of chemicals and fuels imperative. In this sense the use of biomass represents an excellent alternative due to its renewable nature and wide availability. The strong dependence on fossil fuels for the production of both energy and chemicals is one of the main environmental and political concerns worldwide. Even though some several renewable energy technologies are currently under development or already available, such as solar or wind energy, for the production of fuels and chemicals biomass seems to be the only feasible renewable source due to its high carbon content. Biomass is formed by cellulose, hemicellulose and lignin, biopolymers that have a great potential for the production of valuable chemicals.
Those plants, however, which are most easily converted into fuel and chemicals, are food plants. In order to avoid using food as fuel, the focus has shifted to the biogenic
part contained in the municipal solid waste (MSW). This also imparts other advantages such as the valorization of the waste, and reducing its socio-economic cost and environmental impact. Valorization of the biomass contained in the MSW comprises several steps that include both thermo-mechanical and biodegradation processes. However, the product resulting from the biodegradation process is characterized by a wide range of molecular weights. Therefore, this material needs to be fractionated and purified in order to obtain several fractions of narrower molecular weight ranges that can be used as feed-stocks in the industry or as starting materials for the production of other chemicals such as surfactants. FeyeCon Group (FeyeCon D&I and Separex SAS), within the Waste2go project, has investigated and developed four different technologies in order to fractionate and purify the cellulosic material obtained after the biodegradation treatment of MSW. All these technologies were first prototyped and tested by using α-cellulose as substrate
in order to set-up the methods. Once their suitability for cellulose fractionation was proven, the same technologies were used to treat the MSW material and produce different fractions with different potential.
Three of these technologies are based on solvent solutions: metal complexes (LiCl/DMAc), ionic liquids, and aqueous alkali metal hydroxide solutions. These systems are known to dissolve cellulose, which is sparingly soluble in most common solvents. However, just dissolving the cellulose is not enough. In addition it is necessary to develop the process in such a way that different fractions with different molecular weight ranges can be obtained by varying certain key parameters. Mixtures of lithium chloride in dimethymacteamide (LiCl/DMAc) have been extensively reported in the literature as suitable solvents for cellulose with the concentration of LiCl being the limiting parameter to control cellulose dissolution at a given temperature. We were able to show that higher concentrations of LiCl, the longer cellulosic chains. Due to this, it is possible to obtain different fractions of different average degrees of polymerization (DP) by consecutive dissolution of the cellulosic material in LiCl/DMAc solutions containing increasing amounts of the salt.
Aqueous sodium hydroxide solutions are also suitable solvents for cellulose when the concentration of NaOH lies within a certain range. The other key parameter that
influences the polymer solubility in these solvent mixtures is the temperature: low temperatures are required for dissolution since at room temperature cellulose does not dissolve but swells. By varying the dissolution temperature it was possible to obtain several fractions of different DP from the same raw material. Pure aqueous NaOH was found to be a poor solvent for cellulosic materials; however, additives such as urea and thiourea enhance the solubility leading to higher yields. There is a wide variety of ionic liquids that can dissolve cellulose successfully. However, dissolution of the polymer in these solvents will just lead to a negligible decrease in the DP of the polymer. In order to depolymerize the cellulose an acidic catalyst needs to be added to the mixture. The catalyst and ionic liquid employed in this technology were selected jointly taking into account several limiting factors such as the viscosity and melting point of the ionic liquid, solubility of the cellulose in the ionic liquid and stability of the catalyst in the solvent. On the other hand the catalyst should be heterogeneous, so as to avoid tedious separation processes, and should possess a macro porous structure with a large surface area in order to overcome the mass transfer limitations arising from the high viscosity of the mixture. Fractions containing different average degree of polymerization could be obtained by varying the reaction time. The longer the polymer is in contact with the catalyst the further depolymerization proceeds and, therefore, the lower the molecular weight present in the sample obtained.
The forth technology that has been developed for cellulose fractionation is super- or subcritical water. Supercritical water, that is water at a temperature and pressure above its critical point (374.1°C and 22.1MPa) represents a promising alternative for cellulose valorization due to its excellent solvation properties for organic compounds as well as the fact that it can act itself as an acid catalyst thus depolymerizing he cellulose without the need of another catalyst. However, the reaction is difficult to control. It is extremely fast, and often results in the total degradation of the substrate. Also, the fractionation in supercritical water requires specific equipment that has been designed and built specifically for this project by Separex SAS.
Fractionation of α-cellulose was carried out at supercritical conditions leading to the total degradation of the polymer at already one minute reaction time. Due to this it was decided to use subcritical conditions instead. Subcritical water, liquid water at temperatures between the atmospheric boiling point and the critical temperature, shares many characteristics that make subcritical water a suitable for cellulose depolymerization (it is a good solvent for cellulose and can act as an acid catalyst), however, the reaction speed is lower in this case, which makes the reaction easier to control.
Different types of feeds were treated at subcritical conditions: α-cellulose, TM-MSW
(thermo-mechanically treated but non-biodegraded material), and TM–MDSW-D (thermo-mechanically and enzymatically degraded material). In the three cases the treatment with water under subcritical conditions led to the decrease of the average DP of the materials and the production of cellodextrin mixtures. This technique is very innovative and brings two important advantages for the valorization of the municipal solid waste: it does not require a costly pre-drying step and it can lead to the destruction of non-desirable compounds that might be present in the waste material.
Potential Impact:
Life cycle assessment (lca) is an ISO 14040/44 standardised method to assess the environmental impact of products, processes and services along their value chain including the production, the use and the end of life phase. The lca method and the gained results support decision makers like politicians, individuals, and engineers to include environmental aspects during different life cycle phases and to identify and then minimize potential shift of burdens.
To determine the environmental impact of first, the individual processes of the Waste2Go approach and second, the Waste2Go approach itself, a systematic collection of all mass and energy flows has been done. The flows were confirmed and quantified by the project partners, transferred into the GaBi software and the environmental impact calculated. Subsequent the process with the highest significant environmental impact was determined. By means of this information engineers could
include environmental aspects early in the development phase to redesign ecological drivers to minimize the environmental impact.
The enzyme production process was calculated to have the highest environmental impact of all of the treatment steps employed accounting for roughly 70% of the total environmental impact of the process across a number of measurements. Based on the actual state of the technology development upscaling scenarios were performed for the enzyme production, the biodegradation process and the subcritical treatment.
Based on the upscaling assumptions it would possible to reduce a huge amount of CO2-equivalents emitted by the Waste2Go approach. It is worth to concentrate on the reduction of the energy demand and to reuse the enzymes as they are the main drivers. Especially the reusing of the enzymes shows a high potential to reduce environmental impacts. This is explained by the significance of the enzyme production process.
Dissemination and exploitation
Work was undertaken to facilitate the impact of the research outputs in its commercial exploitation by duly protecting intellectual property. The partners joined this project to develop the technology and to commercially benefit from its wider uptake and commercialisation so were therefore actively involved in dissemination activities. Whilst due to budgetary constraints the allocation of person-months per participant in the request for funding from the EU had been restricted, the partners all contributed additional resources at their own expense to ensure maximum impact.
As part of the dissemination activities it was important to ensure that the protocols developed included ongoing protection of potential foreground IP, to plan for post project exploitation and to undertake dissemination actions that enable the partners to maximise the commercialisation of the project results. The following activities were planned for the project lifetime:
• Three international seminars with a total audience in excess of one hundred and fifty attendees
• The submission of more than ten articles for publication
• More than two hundred and fifty visits to the public facing pages of the project web site and
Dissemination and communication activities in Waste2Go commenced from the very beginning of the project to raise awareness of the target groups on the objectives and the upcoming activities of the project from which they could benefit, as well as to engage them in those activities. A visual identity for Waste2Go was developed right after the project commenced and related graphical elements were integrated into the specific dissemination and communication tools.:
The Dissemination Protocols for the project were developed at the early stage of the project as an intra-consortium document, has served as guidance for the Waste2Go partners to conduct more effectively the dissemination and communication activities.
Various means and tools of dissemination and communication have been developed as part of the Dissemination and Exploitation work package in Waste2Go. The following specific means and tools have been conceived and utilised by the partners:
• Project homepage (Waste2Go website) – www.waste2go.eu
• Project brochure
• Project poster
The Waste2Go Website
The Waste2Go website (www.Waste2Go.eu) was launched in January 2013, shortly after the project’s kick-off meeting and has been online for over 38 months. It provides information about the objectives, activities and expected results of the project, presents the project partners, and includes News and Downloads areas that had been duly updated to facilitate the visitors with the latest news and publicly available documents and deliverables generated by the project activities.
Web statistics help to better understand user behaviour and fields of interest when visiting the website, thus facilitating more focussed dissemination and content development. The following graphs indicate some basic statistics regarding the hits on the project website since its launch and concluding at closure.
Total number of visits at Waste2Go’s homepage since the launch of the site in April 2013 reached beyond 5800+ of which around 4100+ are unique visitors (exceeding by far the initially planned visits to the site). The value of unique visitors refers to the number of different IP addresses which were used to visit the Waste2Go web site, while the number of visits describes the total number of visits which were carried out in the given period including multiple visits from the same IP address. The pages value explains the total number of opened sub-sites within waste2go.eu while the bandwidth refers to the total amount of data which was downloaded from the site, including the available deliverables, brochures, etc.
The project’s outreach wasn't limited to the participating countries, but also attracted some interest towards its activities at other EU countries as well as at countries beyond the EU, most likely due to the fact that some high profile international conferences were attended by the representatives of the consortium and their contribution initiated interest towards the Waste2Go activities.
Waste2Go brochure
A project brochure (electronic and hardcopy) was produced right after the project started and was distributed at relevant events that were attended during the project duration by the partners and is available at the public section of the project website. It contains the overall aim of the project and the actions that are undertaken to achieve this aim, as well as the results from the completed WPs and the expected outcomes from WPs. It also contains a link to the homepage of Waste2Go, the general inquiry e-mail address as well as the contact details of the partners.
Waste2Go Poster
There were two different versions of the poster created for two distinct purposes one to provide a more general overview of the project’s activities and the other being more specific about the Life Cycle Assessment activities (related to the project’s sustainability work package). Following this approach there were a number of other poster versions generated primarily by Geonardo to suit the different needs of those partners who were participating at international or local events. These posters helped raise awareness of the project and its activities at a number of high profile events such as at the “Research & Innovation for a circular economy in European Regions”.event in Brussles or the New Zealand Life Cycle Assessment Conference 2014 in Wellington, New Zealand, just to name a few. (full the list of dissemination activities please refer to the summary table.
Waste2Go Press releases
The Consortium aimed to inform the wider public about major achievements of the project that were not subject of IP protection . For this reason, 3 set of press releases were drafted and circulated online to raise awareness on the projects activities and to initiate potential future collaborations for the project partners with non consortium enterprises. The first press release was covering the launch of the project and its website and the second focused on the both new hardware and the biomixer that had been developed at CPI within the scope of WP5 activities. The last press release focused on spreading the word about the final industry seminar held in Brussels in September 14, 2015.
Waste2Go on the web
Waste2Go partners were active in disseminating and promoting the project as a whole and their project-related activities on various online media channels.
Waste2Go Articles for Publication
By the end of the project, the following scientific papers were submitted by the partners on the project findings. Please note that some of these manuscripts will have been published after M36 but they were either approved by the journal or at least they had been submitted for review before the project ended.
• Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase. Biely P, Cziszárová M, Uhliariková I, Agger JW, Li XL, Eijsink VG, Westereng B. Biochim Biophys Acta. 2013 Jul 24; 1830(11):5075-5086.
• Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose. Peter Biely , Mária Cziszárová , Jane W. Agger , Xin-Liang Li , Vladimír Puchart , Mária Vršanská , Vincent G.H. Eijsink , Bjorge Westereng. Biochim Biophys Acta. 2014 Jan;1840(1):516-25.
• Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. J. W. Agger , T. Isaksen , A. Varnai , S. Vidal-Melgosa , W. G. T. Willats , R. Ludwig , S. J. Horn , V. G. H. Eijsink , B. Westereng. Proc Natl Acad Sci U S A. 2014 Apr 29;111(17):6287-92.
• Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Anikó Várnai , Campbell Tang , Oskar Bengtsson , Andrew Atterton , Geir Mathiesen , Vincent GH Eijsink. Microb Cell Fact. 2014 Apr 18;13(1):57.
• A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Stjepan Krešimir Kračun , Julia Schückel , Bjørge Westereng , Lisbeth Garbrecht Thygesen , Rune Nygaard Monrad , Vincent G H Eijsink, William George Tycho Willats. Biotechnol Biofuels. 2015 Apr 23;8:70.
• Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite -1. Tao Feng, Kok-Phen Yan, Maria D. Mikkelsen, Anne S. Meyer , Henk A. Schols, Bjørge Westereng, Jørn D. Mikkelsen. Appl Microbiol Biotechnol. 2014 Dec;98(23):9667-79
• Recent Progress in Understanding the Mode of Action of Acetylxylan Esterases. – NMBU - Article citation: J. Appl. Glycosci., 61, 35-44 (2014).
• The effect of paper source and pretreatment conditions on the enzymatic degradability of waste paper to mono- and oligosaccharides. – NMBU – Manuscript in preparation
• Biosurfactant production from a xyloglucan-derived oligosaccharide. – NMBU – Manuscript in preparation
• Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. – NMBU – submitted for publication
• Comparison of the roles of endoglucanases and LPMOs in the liquefaction of waste paper. – NMBU – Final data acquisition is on-going to finalise and submit the manuscript.
• Comparison of four methods to fractionate cellulose based on degree of polymerization. – NMBU – Final data acquisition is on-going to finalise and submit the manuscript.
Dissemination to the wider public
Following internal publication of the results, protection of all Foreground IP and the publication of scientific papers, the partners aimed to disseminate the Waste2Go project results to the wider public through the internet, TV, newspapers, radio, the popular scientific press. Each partner was be responsible for identifying opportunities for dissemination at a local, regional or national level and advising Geonardo, the WP leader, of these opportunities which would then be reviewed for implementation by the PSC.
Despite the highly technical nature of the core of research within the Waste2Go consortium, with special attention NMBU were able to deliver the message to the wider public in a fashion that was comprehensive for the non-specialist target group as well. The staff carried out a number of activities targeting the non-specialist public to ensure that the goals of the project reached beyond the ears of the scientific community. For this reason the NMBU crew gave a number of oral presentations targeting the policy makers and the general public and published articles on national level press:
• 03.04.2014 Vincent Eijsink; Enzymteknologi for norsk bioraffinering (NorZymeD); invitert foredrag på kontaktseminar mellom TINE og forskningsmiljøer; Olso, Norge. (Presentations in Norwegian for the Norwegian dairy industry; appr. 30 attendants)
• 04.04.2014 Vincent Eijsink; Mulige og aktuelle områder for FoU for realisering av skogens potensialer; foredrag på et lokalt møte om SKOG22 prosessen. Ås, Norge. (Presentation in Norwegian on the valorisation of forest resources)
• 22.05.2014 Vincent Eijsink, Nye enzymer for å utnytte det grønne karbonet; invited lecture in Norwegian at the 2014 «Energikonferansen», Oslo, Norway. Approximately 200 participants from research institutions, companies and governmental organizations, including high-level politicians.
• 26.05.2014 Vincent Eijsink, Using enzymes for valorizing biomass; an overview of the NorZymeD project and enzyme applications in biorefining. Invited lecture at the annual seminar of the Cycle project, Hell, Norway. Appr. 40 participants from research institutions, companies and governmental waste processing organizations.
• 30.10.2014 Vincent Eijsink, «Enzymteknologi i bioraffinering – Muligheter for fremtidig verdiskapning»; Presentasjon for lederen i SKOG22, Ås, Norway, Oct 30, 2014. (Presentation in Norwegian for the leader of a Governmental group working of the valorization of forest resources).
• 28.04.2014; I fremtiden spiser vi trær; NMBU webpages (Bjørge Westereng).
• 05.05.2014; I framtiden kan vi ha trær på menyen; article in Nasjonen (national newspaper) (Bjørge Westereng).
• 06.05.2014: I fremtiden spiser vi trær; article in Dagsavisen (national newspaper) (Bjørge Westereng).
Waste2Go also released a one page project introduction in the Parliament Magazine’s GreenWEEK edition in June 2015. The Parliament Magazine is the policy magazine of the European Union acting also as the Media for the whole of GreenWeek 2015, Europe’s biggest and most important environmental policy conference. The Magazine was distributed within the European parliament thus reaching all MEPs and political groups, EU council presidency officials, Council of ministers, senior officials; European commission, all EU commissioners and chefs de cabinet; Economic and Social Committee, some members in Brussels; Committee of the Regions, Brussels secretariat; European Court of Justice, senior officials; European Investment Bank, senior officials . Over 50,000 copies were distributed in total.
The link to the Waste2Go article can be located here:
https://www.theparliamentmagazine.eu/articles/magazines/issue-413-1-june-2015
The overall effectiveness and impact of the dissemination and communication activities in Waste2Go was considered to be both of a high standard and effective for the three-year duration of the project. Partners’ activities appropriately covered regional dissemination potential in terms of conferences participation, project promotion to the target groups and liaison with other relevant stakeholders. For more details, please refer to Annex E of this report.
The project’s international exposure reached the high standards set prior to the launch of the initiative. The international conferences significantly contributed to the website visits from non-European countries. In line with mid-term expectations, the national and international coverage of the project became more frequent as a result of both increased project activity and the delivery of cutting edge scientific results.
The partners were active throughout the project, attending national, regional, European even oversea events to promote Waste2Go and its activities to the relevant target groups. The second half of the project saw a more targeted dissemination and communication by all partners. The table In Annex E provides an exhaustive list of list events that Waste2Go partners attended over the whole duration of the project.
List of Websites:
http://www.waste2go.eu/