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Biotechnological conversion of carbon containing wastes for eco-efficient production of high added value products

Final Report Summary - ANIMPOL (Biotechnological conversion of carbon containing wastes for eco-efficient production of high added value products)

Executive summary:

ANIMPOL elaborated an environmentally sound biotechnological process for conversion of residues of the animal-processing industries towards marketable plastic items.

Until the end of the project, specific animal residues of porcine, bovine and avian origin without interference with human nutrition, were allocated and delivered for production of biodiesel by advanced catalytic methods. Catalytic transformations of this biodiesel by cracking and isomerization to yield more odd numbered carbon chains were a major task of the final project phase. Detailed information was pulled together relating to biodiesel production and rendering for the economic model for assessing feasibility. Other technical aspects regarding the combustion of distillate, effluent treatment, and others, were also considered. In addition, an appraisal of the saturated biodiesel market was carried out which considered changes that might impact on the economics of the ANIMPOL process if realized on an industrial scale.

Project context and objectives:

Specific objectives of the project and how they were addressed during the project period

- Design of an integrated industrial process dedicated to the bio-mediated, cost-efficient production of value-added, biodegradable polyhydroxyalkanoate (PHA) biopolyesters, by starting from lipid waste (tallow) from slaughterhouses, rendering industry, and waste fractions of the biodiesel production. These wastes are upgraded to the role of resources for renewable raw materials, thus bringing together diverse waste producing players.

This envisaged process was designed based on the experimental input of all project partners. A detailed and complete process flow diagram, encompassing energy and material flows and balances, was elaborated and is available for industrial implementation.

- Improvement of the quality of biodiesel by removal of its saturated fraction.

The saturated fraction was removed from the animal-based biodiesel mix by a convenient technique and successfully applied as fermentative carbon feedstock. In addition, this saturated fraction was used for catalytic generation of precursors for biotechnological production of special PHA-building blocks (3-hydroxyvalerate).

- Assessment of the raw material sources (lipids from animal waste, saturated biodiesel fraction, surplus glycerol from the biodiesel production) for the fermentation process by selected microbial strains suitable for the production of structurally diversified PHAs under physiological stress conditions.

Different waste fractions were investigated regarding the fatty acid pattern of their lipid fraction. The lipids were converted to biodiesel which, together with glycerol as the second waste stream of the lipid conversion, was used as carbon source for various microbial PHA producers.

- Aiming at the improvement of microbial growth on the selected substrates and the quality and amount of the polymer produced; appropriate strains were studied by different approaches which include recombinant gene expression and host cell genome modification for enhanced expression of nuclease and deletion of intracellular PHA breakdown.

Strains were selected and assessed both for the production of thermoplastic scl-PHA and latex-like mcl-PHA.

- Set-up of microbial growth and production phase amenable to be scaled-up for a novel optimized directed production of structurally predefined PHAs. The experimental polymer production under efficient control strategies using fed-batch cultivation will be developed aiming at reproducible product quality. Predefinition of the polymer structure on a molecular level, hence the monomeric composition was accomplished by triggering the feeding of main- and co-carbon sources.

Reproducible fermentation protocols for up-scaling the process to industrial conditions were established. The fermentation experiments were kinetically analyzed and converted into mathematical models.

- Development of an environmentally safe and efficient downstream processing for recovery and purification of PHAs; separation of PHAs from microbial biomass by cheap and ecologically benign techniques avoiding harmful solvents, focusing on mechanical cell disruption techniques, were implemented, monitored, and assessed.

Novel recovery methods with restricted solvent requirements were assessed and compared to classical PHA extraction methods.

- Chemical, structural, biological, physical and mechanical characterization of the produced PHAs.

The biotechnologically produced materials were characterized regarding their material properties. New dependencies of the cultivation conditions on the material properties were recognized and described.

- Preparation of blends and composites of the PHAs with selected polymeric materials including synthetic analogues of PHA, inorganic and/or organic fillers like nanofillers. The organic fillers will be taken from renewable agro-waste (lignocelluloses, polysaccharides and surplus crops), either directly or after appropriate physical or chemical modification whereas organophilized inorganic fillers (clays, sepiolithes and calcium carbonate) will be used.

Blends and composites were produced based on PHA and various compatible materials (clays, hazelnut shells, pine shells, natural fibers etc.) and characterized.

- Engineering design of PHA production, extraction and purification unit operations combined with the analysis of the cost effectiveness of the industrial process as set up in the down-stream processing of slaughterhouses, medium and large rendering and biodiesel factories.

The process was designed based on the facility units and is ready for industrial realization.

- The cost effectiveness of the process for industrial scale production of PHAs will be assessed in terms of costs of raw materials, chemicals (additives, cofactors, solvents) and energy required for the production of PHAs and its blends. This issue is a key factor for the success of the project as the introduction and penetration into the market of new polymeric materials has to be competitive in terms of cost/performance balance.

Project context considering ecological and economical aspects

Based on ecological considerations, an increasing need for industry to switch from classical plastic materials to biobased alternatives like polyhydroxyalkanoates (PHAs) is nowadays undisputed. This can be underlined by the estimated amounts of about 280 Mtons of petrochemical plastics produced globally in 2013, with expected annual increases of 4%. Economic reasons are generally considered as the major obstacles for replacing conventional commodity and semi-commodity plastics by PHAs on a relevant scale. The project was needed as a viable strategy to enable production of these highly promising biopolyesters in Europe, to avoid that in the future PHAs have to be imported from outside the EU. If successfully implemented now, these seminal biopolymers will be decoupled from manufacturing in various global regions, where biopolyester and biofuel synthesis competes with human nutrition, and frequently lacks consideration of environmental requirements and acceptable human working conditions.

Specific background regarding raw materials and products

Only a few technologies are found in current industrial processes that can successfully convert feedstocks that are made up of a high percentage of saturated fat. The most successful technology which has been developed and implemented is the production of fatty acid methyl esters (FAME) that can be used as liquid fuel (biodiesel). The most prevalent issue with FAMEs is its operability at lower temperatures specifically with cold filter plugging (CFPP) point at blends over 20% saturated FAMEs on vol/vol basis. Considering blends greater than 20% would require modifications to the vehicle injection system. An operator that uses a saturated feedstock also requires a good understanding of handling as the raw material often solidifies at room temperature.

PHAs constitute a family of polyesters classified as bio-based, biodegradable and biocompatible materials. The spectrum of potential applications ranges from simple packaging materials featuring advantageous properties like a high oxygen barrier and UV-resistance, to high-quality materials to be used in special niches, e.g. in the medical and pharmaceutical field. Economic reasons are pointed out as the major obstacles for replacing common plastics by PHAs on a relevant scale.

Plastics that should be replaced by novel materials like PHA are those accruing at huge amounts, such as polypropylene (PP) or polyethylene (PE). These materials are utilized only during a relatively short time span. After that, they are often incinerated, elevating the atmospheric CO2 concentration. The main problem arising from incineration of plastics is the same as for energy recovery from fossil feedstocks: carbon that was fixed during millions of years and that within this time was not part of the natural carbon cycle, is converted to Carbon dioxide (CO2) that can accumulate in the atmosphere, contributing to negative climatic effects.

Beside, more and more waste of the said highly recalcitrant plastics that are not incinerated is piled up every year, thus aggravating the landfill crisis. Recycling systems demand a sufficient degree of purity and a certain sorting accuracy. In addition, the collection costs are fairly high, and each recycling cycle has a negative impact on the mechanical properties of the materials, such as an increase in brittleness.

In order to become competitive on the market, the price per kg of a biopolymer for a special application must be in the same range as the competing 'classical' plastic. Hence, the production costs of PHAs have to be minimized considerably despite the increasing market price for mineral oil. According to the 'state of the art', these biopolyesters are produced on a larger scale starting from rather costly carbon sources like purified sugars. Classically, it is estimated that the PHA production costs from purified substrates are about five to ten times higher than the costs for petrochemically produced plastics like PP or PE, as indicated by the market prices for PHAs from China (companies Tianan, Tianjin and DSM), the USA (Telles, Tepha Inc., P &G), or Brazil (PHB Industrial S/A); this goes for the case of thermoplastic scl-PHAs. In the case of mcl-PHA to be used of elastomers, the market prices are even higher (Polyferm Canada; about 200 US-dollars per 10g of mcl-PHA).

On the one hand, recent studies indicate that this production of PHAs from such 'classical' carbon substrates has been economically optimized to a high extent. On the other hand, diverting materials that are of high importance for nutrition of mankind, towards biotechnological production of 'green plastics' raises serious ethical conflicts considering the enormous number of starving people worldwide and the increase in the world population, and hence in needs for food and commodities. Converting nutrients like edible oils or sugars to fuels or polymers cannot be regarded as an ethically viable strategy for the future (see contemporary 'food versus fuel' or 'plate versus plastic' controversy). In contrast, the utilization of diverse waste streams which do not feature any value for nutrition but constitute severe disposal problems for the industrial branches where they accrue can be considered as the most promising approach in making PHAs more competitive. In the past, successful attempts in utilizing waste materials for PHA production have been done based on surplus whey from dairy industry (WHEYPOL, Growth EU project GRD2-2000-30385) and more recently from wastewaters and perujo of olive oil pressing industrial units. (POLYVERY Graft FP6 EU Project COOP-CT-2006-032967). In the case of PHA production from whey, calculations show that it was possible to lower the production price to below 3 EUROS per kg pf PHA. In the proposed project, waste streams from slaughterhouses, rendering industry, and waste fractions of the biodiesel production are upgraded to feed stocks for biotechnological PHA production. These waste materials constitute innovative alternatives to the conventionally used carbon-sources and processes. The proposed utilization of the waste resources mentioned above contribute to the reduction of the production costs and feature a future-oriented approach of solving disposal problems for the involved industrial branches.

Biotechnological PHA production occurs in aerobic processes, therefore only about 50% of the 'classic' carbon sources like sugars, and even a lower percentage of the precursors used for generation of copolyesters end up in the desired products. Normally, 0.48 g poly-3-hydroxybutyrate (PHB) can be expected per g sugar (value without taking into account the sugar consumption for biomass formation). Based on sugar cane sucrose, the company PHB Industrial S/A in Brazil produces, on pilot scale, PHB at a mass conversion yield of 0.3 g/g. Based on the metabolic circumstances of the microbial PHA producers (beta-oxidation pathway for break-down of long alkyl chains to acetyl-CoA), this value can be doubled using biodiesel instead of carbohydrates like sugars. This was demonstrated by the results obtained in ANIMPOL.

During the last couple of years, the preparation of composites and blends has turned into one of the key research fields in biopolymer science. For enhancement of the material properties, PHAs or their derivatives like polyester urethanes can be processed together with a variety of compatible matters, resulting in the creation of novel PHA-based blends and composites. For this purpose, the utilization of polymeric materials like poly(vinyl alcohol) (PVA), PLA, poly(e-caprolactone) (PCL) etc., including synthetic analogues of PHA (e.g. atactic PHB), inorganic fillers (clay materials or calcium carbonate), and organic fillers of agricultural origin such as bagasse, powdered or in form of fibers, was successfully tested. Concerning fillers from agriculture, the application of surplus materials like lignocelluloses such as sugar cane bagasse, hazelnut shells, wheat flour, fruit peels, crop fruit fibers, saw dust, wheat straw, or cellulose derivatives (e.g. cellulose acetate) is of major interest.

In general, nano-composites and natural fibers composites have to be distinguished. Nano-composites have the potential to enhance special polymer properties, such as gas permeability and thermal and mechanical characteristics. Novel nano-biostructured packaging materials are described especially for application in the food packaging sector. For creation of nano-composites, rather small amounts of the filler, usually an organophilic modified clay, are needed for efficient enhancement of the material performance. Natural fibers composites often display excellent mechanical properties, and, as desired for many applications, they result in a lower density of the final product. Due to the fact that in most cases fibers constituting agricultural residues are used as fillers, the biodegradability of the final product is generally enhanced. Because such fibers do not feature a considerable price or even constitute waste materials, this normally goes in parallel with a reduction of the entire production cost of the marketable composite product. Therefore, ANIMPOL developed also a process for using agricultural waste as filler for production of PHA-based composites. This provides an additional solution for agriculture to upgrade surplus materials, and further lowers the overall costs of bioplastic items.

Project results:

The following section provides a summary of S&T results/foregrounds and details for each task in line with the structure of Annex I to the Grant Agreement on a WP and task-by-task basis; significant results are highlighted

WP1: feedstock and upstream processing (RDT)

Task 1.1: supply and costing of raw materials

This task is dedicated to the supply of raw materials and the examination of sources of raw materials. The deliverable of all raw materials was accompanied by the examination of the related costs. The allocation of animal waste was done by Partner 11 (RIX) and the saturated biodiesel fraction was delivered by Partner 5 (ARGENT).

A broad range of animal waste materials, saturated FAME (biodiesel) and its by-product crude glycerol phase was delivered by the beneficiaries RIX and ARGENT since the beginning of the project. The delivery occurred according to the requirements of the project partners doing further work with these materials (TUG, UNIPD, KFU) for other work packages. These requirements and allocation strategies for special waste fractions (e.g. porcine) where discussed during several direct meetings and via means of telecommunication. It can be underlined that the flow of raw material during the entire project period was fulfilling the needs for all involved project partners.

The responsible company RIX delivered the subsequent materials to the before mentioned project partners:

January 2010 (to get started!):

3.7 kg of pork belly with rind (residue from the bacon production) as lipid-rich reference material. Material analyzed in details by partner KFU

Delivered later during the first 6 months:

- Entire amount of more than 10 kg of 4 different porcine organs that clearly constitute waste materials for the meat processing industry without interference with food or feed purposes (intestines and guts, udder, spleen, heart)!!

Delivered later:

-High quantities (22 kg) of a variety of special organs and waste fractions (e.g. porcine liver with high amounts of odd-numbered fatty acids, porcine reticulum, lungs, epididymis [fatty acid pattern was unknown before!]

-Ca. 5 kg of chicken waste. The company optimized crushing of poultry bones by milling prior to drying via lyophilization.

-Crushed bovine head (including brain; crushing procedure optimized in the company; 17 kg)

-Bovine spleen (8 kg)

As an additional task, the company RIX contributed to the cost assessment of the materials.

Materials displaying a market value [EUROS/kg]:

Heart: 0.85

Lung: 0.60

Liver: 0.70

Spleen: 0.80 (Bovine: 0.70)

Reticulum: 0.60

Udder: 0.40

Materials that have to be disposed of (cost demanding!):

Porcine testicles: disposal 0.30 EUROS/kg.

Bovine residues: Spinal marrow, intestines and guts, Head plus brain: risk materials – disposal 60 EUROS/t.

Avian residues (poultry waste): real waste material normally to be disposed of!

Since the start of the ANIMPOL project there have been a number of significant changes in the marketplace. These changes will impact directly on the economics of the overall process especially when considering the overall production costs in the generation of PHA's from the saturated fraction of animal fats. The original idea behind the project was to split the saturated fraction from that of the unsaturated part. The saturated fraction deemed as the inferior part would be used in the PHA production process using biotechnology. The unsaturated fraction seen as the high quality part of the methyl ester would be used as a high quality fuel. There was also a more desired fraction which possesses odd number carbon chains that would add further value the final biodegradable plastic.

When tallow derived methyl ester was first introduced into the market overall volumes were scares as this type of material was not fully understood. This made the trading of this material somewhat problematic. The blenders and fuel distributors were nervous of utilizing products that possess higher levels of saturation as this was seen as inferior to the unsaturated vegetable based oil. Their concerns were focused to operational issues in the field, especially those related to the temperature and more specifically cold flow plugging point (waxing/crystallization of fuel).

Argent has been supplying the market since 2005 with both used cooking oil (UCOME) and tallow methyl esters (TME). Initially supplying a higher proportion of UCOME this has now changed to predominantly category I tallow. During this period Argent secured a route for a mix of UCOME and TME to a major distributed/ petrochemical producer this was the first major step to instill a degree of confidence. This continued over a period but never saw dramatic increases of TME being blended into main stream fuels. Argent, were then able to supply TME to a major global producer/distributor that were confident that this product could at least be utilized over the summer operating period. This continued for a lengthy period until economics dictated that we change. At this period Argent were seeing increase selling prices, however the differential between vegetable based fuels and TME were still sizeable.

Increased lobbying and the participation from Argent employee/experts over the years to the present date have increased the awareness and the acceptance of more saturated products in the marketplace. Involvement/participation in the construction of new fuel standards both on a national and European basis how facilitated an increased scope of new products from different raw materials. The new revision of EN 14214 has seen the introduction of a new distilled grade which includes TME with lower saturated mono-glycerides which can be used in a much greater range of blend over winter periods. This is seen as a crucial step for increasing potential volumes of biofuels to the market place. Emphasis is always focused around quality and potential operating constraints to protect the fuel market. On this basis TME is characterized a fuel of the highest quality which is fit for purpose which is significant change in classification from a number of years ago when it was classified as inferior. TME in its saturated form has some major advantages over the non-saturated types in that the product is more stable and it possess a higher cetane numbers that improves combustibility. The cold climatic using tallow can also be met under the current mandate of a 7% biofuels inclusion into a good quality petroleum base diesel.

The use of TME has also found new applications in higher blends that go much further than the 7%, in fact Argent currently supply blends in excess of 20%. These blends are used in fleet application like buses and have proven to be highly successful.

Other areas that have further enhanced the profile and value of TME is the introduction of the certificate system for methyl esters that have been generated from waste products like category I tallow and used cooking oils.

The renewable transport fuel obligation (RTFO) order obligates fossil fuel suppliers to produce evidence showing that a percentage of fuels for road transport supplied in the UK come from renewable sources and are sustainable, or that a substitute amount of money is paid. All fuel suppliers who supply at least 450,000 liters of fuel a year are obligated. This includes suppliers of biofuels as well as suppliers of fossil fuel.

Owners of biofuel at the duty point are awarded one (RTFC) per liter of biofuel supplied, however the December 2011 amendment to the RTFO order introduced double rewards for some fuel types, including those made from waste materials such as CAT 1 tallow and UCO, together with a requirement to have data on the carbon and sustainability performance of fuels to be independently verified before renewable transport fuel certificates (RTFCs) are awarded.

RTFCs may be earned irrespective of the volume of biofuel owned, providing a potential revenue stream for even the smallest suppliers. RTFCs may be traded between participants in the scheme.

Currently RTFCs are trading at around 15 EUROS-cents which equates to 30 EUROS-cents per liter or for that biodiesel which is eligible for double counting.

Changes in EU legislation relating to biofuels are also beneficial to further the use of TME i.e. Renewable Energy Directive and possibly ILUC.

The sales differential between TME and more conventional fuels like SME, RME, and UCOME has lessened over the years on the basis that it is produced to the same high quality but also they can operate under regional climatic conditions. Prices a few years ago were as low as 70 EUROS-cents for TME and these have been as high as 97.5 EUROS-cents however this varies somewhat. There is also a differential of approximately 5-6 euro cents between UCOME and tallow however the raw material is higher than that of category I derived TME. It should also be stated that the cost of category I tallow has changed substantially from about 243 EUROS euro/t to 585 EUROS/t and edible grade category III has a premium of about 67 euro/t. As category III tallow is used as a feed this will not be eligible under the certificate system and would forfeit the enhanced value in the biodiesel. These values translated into cost in the manufacturing matrix would have major implications on the viability of the PHA production process.

In addition to the delivery of biodiesel to the involved project partners according to their requirements during the entire project time, the work carried out during the later period investigated catalytic transformations of tallow methyl ester to yield more odd numbered carbon chains. A literature search was first conducted followed by extensive reaction work using numerous catalysts. The material was cracked and isomerized to try to achieve goal. The work was productive in that the amount of C15 and C17 was considerably increased. The solubility of the final was an issue for the fermentation and we looked to find a suitable dispersant.

A lot of separation techniques were investigated with limited success especially in terms of yields and these processes would be very expensive on an industrial scale. Other industrial scale processes were also considered during this investigation.

Detailed information was pulled together relating to biodiesel production and rendering for the University of Graz to use this in their economic model for assessing feasibility. Other technical aspects were also considered and supplied to the University regarding the combustion of distillate and effluent treatment etc.

Finally an appraisal of the saturated methyl ester market was carried out which considered changes that might impact on the economics of the project going forward.

Task 1.2: Assessment of raw materials

Different slaughterhouse waste materials from pork, beef and chicken were delivered from partner 11 (RIX). Out of that, udder, honeycomb stomach, liver, heart, epididymis, testicle, lung, bacon rind, spleen from pork and brain, spleen and head bones from beef and waste material from chicken were extracted, the fatty acid composition was analyzed and the overall quality for the further purpose as a feedstock for transesterification and fermentation was evaluated. All samples were extracted with hexane and the thus obtained fat fraction was determined.

Regarding the search for odd-numbered fatty acids like margaric acid (heptadecanoic acid), acting as 3-hydroxyvalerate (3HV) precursors in biotechnological PHA synthesis, the amount of margaric acid (the expected key co-substrate for 3HV production in the fermentation experiments) was very low. Hence, alternative strategies for production of odd-numbered compounds had to be assessed and carried out (see task 1.3).

Emphasis is put on the monitoring of the different feedstock qualities of waste animal fats and slaughterhouse products. Partner 5 (ARGENT) and Partner 11 (RIX) delivered different feedstocks. ARGENT delivered distilled biodiesel and the corresponding tallow feedstock and RIX animal waste material porcine, bovine and avian origin. The distilled biodiesel was analyzed according to EN 14214. Due to the fact that no specific European standard exists for the determination of the qualities of waste feedstocks for biodiesel production, parameters characteristic in fats and oil analysis where selected and determined. Based on the results a statement about the expected quality of the corresponding fatty acid methyl ester (FAME) can be made. The obtained results are given in detail in Deliverable 1.3 This analytical part was ongoing until the end of the project.

The obtained fatty acid alkyl esters were separated into a saturated and an unsaturated fatty acid fraction via low-temperature-crystallization. The unsaturated fractions had better cold flow properties than their corresponding original esters. For example the cold cold-filter-plugging-point from original fatty acid methyl esters (FAME) decreased from 10 °C to -26 °C in the unsaturated fraction. The different saturated fractions were delivered to partner 1 TUG and investigated as a carbon source for the fermentation process.

Task 1.3: Modification of raw materials

The extracted fat and the tallow feedstock were used for classical and enzymatic catalyzed transesterification reactions. As enzymes two commercial lipases from Candida antarctica and Mucor mihei as well as a sample from Partner 2 (UNIPD) were investigated. For the transesterification experiments methanol, ethanol, propanol, iso-propanol, butanol, tert-butanol and iso-amyl alcohol were used. The overall yields of fatty acid alkyl esters were between 80 – 90 %. The results with tert-butanol were insufficient. Therefore, the production of tert- butyl ester was carried out by the esterification of free fatty acids with isobutene. Also, the ester yields coming from enzymatic transesterification with the enzyme received from partner 2 were insufficiently low.

In addition to the transesterification of animal lipids to fatty acid esters containing odd-numbered alcohol moieties, odd numbered precursors for 3-hydroxyvalerate (3HV) production were produced by chemical catalysis. Here, the saturated fatty acid ethyl ester fraction was taken as starting material for the production of odd-numbered carboxylic acids. This fraction was used to produce fatty alcohol using sodium and abs. ethanol in a first step. Via dehydration at high temperatures and alumina oxide as a catalyst 1-olefins were produced thereof. Finally, a mixture of carboxylic acids (C9 to C17) was synthesized from olefins via oxidative ozonolysis. Also these samples were delivered to TUG and successfully tested as a carbon source for PHA-production.

Task 1.4: Screening and utilization of lipases

Summary: Although Acinetobacter venetianus DSM 23050 lipase activity was found promising using different triglycerides (tributyrin, maize oil, commercial lard, bacon rind or tallow provided by P4) as substrates (both at 37 and 55°C), also other interesting PHA-producing strains were selected: Hydrogenophaga palleroni DSM 63, Pseudomonas oleovorans DSM 1045, Pseudomonas fragi DSM 3456, Cupriavidus necator DSM 545 and Diaphorobacter sp. DSM 13225. More than 50 new isolates were selected for assessing their lipase activity once isolated from environmental samples (water and soil) and from waste (water and mud) coming from a slaughterhouse, and then tested for PHAs production. After an exhaustive discussion with all the Partners a decision was taken to adopt for further studies the two following strains: C.necator DSM 545 and Ps. oleovorans DSM 1045.

UNIPD:

After an exhaustive discussion, the decision was taken to search for bacterial strains, culture collection strains or new isolates that show lipase activity. A bibliographic study was performed in the first period of these 6 last months for the use of bacterial lipases and Acinetobacter venetianus DSM 23050 (or Acinetobacter sp. RAG-1) was selected as promising lipase producer. Other culture collection strains and new isolates from water, soil or slaughterhouse were also studied.

Acinetobacter venetianus DSM 23050: This strain was previously described to produce an extracellular lipase (LipA) with the following characteristics (Snellman et al., 2002 Eur. J. Biochem. 269: 5771-5779): the enzyme LipA is released into the growth medium during the transition to stationary phase, and it was found to be stable at pH 5.8–9.0 with optimal activity at pH 9.0. This lipase remained active at temperatures up to 70° C, with maximal activity observed at 55° C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides.

In the present study Acinetobacter venetianus DSM 23050 lipase activity was tested in solid DSMZ 92 (Trypticase soy yeast extract medium), nutrient or TRA medium containing different lipid substrates for this enzyme. Bacon rind and tallow were delivered by Partner 4. Halo around colonies was detected on solid medium containing different triglycerides (tributyrin, maize oil, commercial lard, bacon rind or tallow), indicating that there is a lipase activity present in all cases. Activity was revealed using the entire culture or only culture supernatants. The signal is stronger when rhodamine dye is used and observations are performed under UV-light.

Lipase activity was also tested in liquid medium containing different substrates for this enzyme. Medium contained triglycerides as carbon sources. Bacon rind and tallow were delivered by partner 4 (KFU). Lipolytic activity was determined at 30 °C, pH 8.0 by spectrophotometric method according to Winkler and Stuckmann (1979, J Bacteriol 138(3):663–670) using p-nitrophenyl palmitate as substrate. The quantitation was carried out with a standard p-nitrophenol curve measuring absorbance at 410 nm. The enzymatic activity (U) was expressed as micromoles of p-nitrophenol released per minute and mL of crude lipase preparation (µmol min-1 ml-1).

Activity was detected in liquid culture in the supernatants of A. venetianus DSM 23050 containing different triglycerides (tributyrin, maize oil, commercial lard, bacon rind or tallow) as substrates.

KFU:

The extracted fat and the tallow feedstock were used for classical and enzymatic catalyzed transesterification reactions. As enzymes two commercial lipases from Candida antarctica and Mucor mihei as well as a sample from partner 2 were investigated. For the transesterification experiments methanol, ethanol, propanol, iso-propanol, butanol, tert-butanol and iso-amyl alcohol were used. The overall yields of fatty acid alkyl esters were between 80 – 90 %. The results with tert-butanol were insufficient. Therefore, the production of tert-butyl ester was carried out by the esterification of free fatty acids with isobutene. Also, the ester yields coming from enzymatic transesterification with the enzyme received from partner 2 were insufficiently low.

Three different lipases were investigated as described subsequently:

Candida Antarctica: The yields of fatty acid alkyl esters were very high with concentrations of about 80 – 90 %. The results using methanol and iso-amyl alcohol showed with 92.3 % and 97.2 % the highest values. The conversion to fatty acid alkyl esters using tert-butanol achieved with 11.3 % the lowest yield.

WP2: Fermentation technology, genetics and mathematical modelling (RDT)

Task 2.1: Production of scl-PHAs

A broad number (about 100) of scl-PHA homo- and copolyesters with different compositions, produced by different feeding strategies and recovered by different techniques, was produced by TUG and UNIPD and send to the responsible partners for characterization. Concerning the co-polyesters, 3HB, 3HV and 4HB building blocks were inserted into the biopolymers. Here, media compositions were optimized, as well as the feeding strategies for the required substrates.

Task 2.2: Production of mcl-PHAs

UNIPD:

A lot of efforts were devoted to the production of mcl-PHAs, mainly using Pseudomonas oleovorans DSM 1045 as suggested by UNIPD according to their research results.

Pseudomonas oleovorans DSM1045 was investigated by as possible mcl–PHA producer in liquid E-medium using different carbon sources (see Tab. 1 of AnnexI-P2 of respective Deliverable 2.7).

A number of PHA samples obtained with the selected strain reported above were sent for chemical-physical analysis to P7, P8 and P9.

TUG

Three Pseudomas putida strains (Ps. putida ATCC29347, Ps. putida KT2442 and Ps. putida DSM1045 [= UNIPD strain Ps. oleovorans]) were analyzed for their ability to grow in the presence of alternative raw materials (biodiesel, gluconic acid and glycerol) and to produce various types of PHAs, constituents of fully degradable bioplastics to be applied as latexes or as special niche products (thermosensitive adhesives, staring materials for postsynthetic modification etc.). In the course of the experiments, different techniques of analytical chemistry (GC, HPLC, etc.) were applied to obtain as valuable and accurate results as possible.

A detailed description of the outcomes using these strains is provided in the subsequent paragraphs:

Ps. putida ATCC 29347

When Ps. putida ATCC29347 was cultivated in glycerol containing medium, the lag phase was shorter than in the case of biodiesel. This finding was independent from the media composition. The faster initial growth with glycerol as a sole carbon source could have been caused by the fact that glycerol has a more simple chemical structure than fatty acids present in biodiesel and, hence, is more easily imported into the cells. Biofuel typically contains up to 14 different fatty acids that were chemically transformed into fatty acid methyl esters. Most of these fatty acids are saturated and thus do not possess any double bonds. Even though the fatty acids are much richer in energy content than glycerol, they require longer metabolic processing than glycerol. This is the main reason why the cell density was increasing rather slowly at initial growth phase. Furthermore, a previous study with various species of Ps. putida growing in FAMEs and glycerol showed that glycerol was the preferred substrate as well as the energetically most favorable substrate for the formation of acetyl-CoA (Silva et al., 2009). Before acetyl-CoA is formed, glycerol is turned into dihydroxyacetone phosphate followed by a conversion to phosphoenolpyruvate and puryvate. Pyruvate is decarboxylated into acetyl-CoA which can then lead to PHA formation. For later growth phase however the cell density in the medium with biofuel equaled or even reached higher value than in the same medium with glycerol.

Ps. putida KT2442

Strain Ps. putida KT2442 was only tested in modified Küng media with gluconic acid as the sole carbon source. As with any other experiment conducted for the purpose of this thesis, two tests were run in parallel so the results could be compared. In this case, the growth patterns in both tests were in good correlation. The cell density increased quite rapidly just like in the case with glycerol mainly due to well-developed cellular tolerance as well as high accessibility by the bacterial cells. The NH4+ concentration was down to zero within 26 h after inoculation suggesting a start of accumulation phase at this point.

Ps. putida DSM1045

Unlike Ps. putida ATCC29347 and Ps. putida KT2440, Ps. putida DSM1045 was grown in the presence of two carbon sources, biofuel and gluconic acid. When cultivated with biofuel as a carbon source, two media compositions were tested to determine which one is more suitable for growth. As for gluconic acid, only modified Küng was applied for the growth.

Task 2.3: Microbiology and genetics

At the 6- and 12-months meetings in Portorož (Slovenia) and in Swierklaniec (Poland), the bacterial strains isolated and/or tested were proposed. A summary with the main information for each strain was designed as reported in previous reports.

Criteria for strain selection were:

No pathogenicity (only risk group I organisms)

Genetic stability

Growth rate on ANIMPOL-substrates

PHA-production rate on ANIMPOL-substrates

Copolyester production possible? (for scl-PHAs)

After an exhaustive discussion with all the Partners a decision was taken to use C. necator as possible scl–PHA producer. C. necator DSM 545 was grown firstly in DSMZ81 solid media using crude glycerol phase (CGP) and biodiesel (both delivered by Partner 5), pure glycerol, stearic acid methyl ester, palmitic acid methyl ester and myristic acid methyl ester, and in triglycerides as bacon rind and tallow (delivered by Partner 4).

Task 2.4: Mathematical modeling and kinetic analysis

Partner 3 (UNZA) have fulfilled their obligation for the ANIMPOL project. During the project time they were successful in modeling and optimization of different types of fermentation performed by C. necator DSM 545 and Pseudomonas chlororaphis DSM 500083 on different substrates (glucose, glucose with glycerol, glycerol, biodiesel, and biodiesel with addition of odd-numbered fatty acids as 3HV-related co-substrate) and different cultivation modes (fed-batch or continuous mode). The feedback of UNZA by the established models help partner TUG to a high extent to improve their fermentation strategies (inoculum preparation, concentrations of nutrients), especially in the case of scl-PHA production by C. necator on the ANIMPOL-related substrates biodiesel and glycerol.

Task 2.5: Preliminary characterization of raw polymers

An exhaustive number of characterization analyses was carried out by the responsible partners for determination of the properties of delivered polymer samples; the results indicate the impact of the different applied PHA production strategies on the PHA quality and properties. In total, 57 samples from TUG, 12 samples from ARGUS and 18 samples from UNIPD were analyzed and characterized.

WP3: Downstream processing, LCA and engineering (RDT)

Task 3.1: Downstream processing

For recovery of PHA, solvent extraction, cell disintegration by ultrasound technique, combined with the separation of released PHA granules via a dissolved air flotation (DAF)-unit, was investigated. Using solvent extraction, focus was devoted to the utilization of non-toxic solvents (Dichloromethane, lactic acid esters, methyl acetate) and in minimizing the solvent quantities. Concerning the classical PHA-solvent chloroform, continuous (Soxleth) extraction was compared with discontinuous extraction (batch). Different pre-treatment methods for PHA-containing biomass regarding the removal of lipids were assessed. In addition, non-solvent cell-disruption using SDS was optimized for scl-PHA containing biomass. All details are available in Deliverable 3.9 and in the experimental part of the Deliverable M.20.

Task 3.2: LCA and cleaner production studies

In this study SPI methodology was the preferred life cycle impact assessment (LCA) method. It results in an ecological footprint, calculating the area necessary to embed the whole life cycle to provide products or services sustainably into the ecosphere. The sustainable process index (SPI) is based on the assumption that the only energetic income of our planet is solar energy. This income drives all natural processes and global material cycles (e.g. the global carbon cycle). The key resource to transform this income into utilisable material (e.g. biomass) or energy is area, e.g. using the techniques like photovoltaic, thermal solar energy or the indirect utilization of solar energy via conversion of biomass. Productive land, air and water have to be retained in a condition that allows them to remain the key production factors in a sustainable economy, therefore all emissions into the three compartments air, water, and soil are considered for the ecological footprint calculation following the principles that global material cycles must not be changed and that the local qualities of these compartments must not be changed either. Therefore the SPI value is a sum of seven different sub-areas (area for land occupation, area for non-renewable material, area for renewable material, area for fossil carbon, area for emissions to soil, water and air).

Details:

Animal residues:

As explained in the economic analysis (deliverable D 3.23) it is assumed that waste cost is equivalent to transportation cost for material collection from different slaughtering facilities. Similarly waste materials from slaughterhouses are considered with an SPI value of 0 m²/t. This allocation is made because the footprint is already included in the main product of slaughtering which is meat. Nevertheless, the transportation of the waste material to the rendering plant is taken into account. For 1 t of PHA production 13.64 t of waste material will be transported within 75 km radius causing 150 km distance per trip. The total freight transportation amounts to 2,046 tkm. The SPI value for transportation using 28 t transportation trucks is 84.95 m2/tkm. Thus the calculated SPI value for waste is 173,855 m2.

Hydrolysis:

SPI for offal hydrolysis is the sum of calculated SPI values for offal transportation, electricity consumption for chopping and acid reclamation, heat consumption for heating and acid consumption. The Ecological assessment result for hydrolysis carried out using SPI Methodology using inventory inputs data for 1 t equivalent of organic nitrogen production through offal hydrolysis. It turned out that reveals that mineral acid and base are the key footprint producers and contribute to about 83 % to the overall footprint of the sub process, while transportation and energy sectors also have significant shares. The higher share of mineral acid and base footprint is due to fossil fuel based highly energy intensive production processes of these chemicals.

Rendering:

SPI calculations for the rendering process are also divided into two parts depending on the material to be processed. Contaminated waste material is exclusively used for heat production for the Rendering II process. In contrast Rendering II processes the main part of the waste stream to produce meat and bone meal (MBM) and tallow.

Biodiesel production:

Biodiesel is produced by transesterification of fat with methanol. Inventory data for 1 t of biodiesel production are provided in D 3.23 and indicate that tallow obtained from rendering process, KOH, H2SO4, CH3OH, heat, electricity and waste water are main energy and mass flows for the process. The raw material (fat from rendering process) along with methanol and electricity are the main contributors to the overall footprint of the process. Fat production is a highly energy intensive process along with being the main material input for biodiesel process accounting to 1.02 t/t biodiesel production.

PHA production:

PHA production, including the downstream processing is comprised of fermentation process, PHA separation and the purification process. Inventory data for 1 t of PHA production in fermentation process are provided in D 3.23. Hydrolysate is a source of organic nitrogen and mixture of essential amino acids while ammonium hydroxide serves as a source of inorganic nitrogen and also helps to maintain reaction conditions. Biodiesel and glycerol are the main raw materials acting as carbon source for bacteria to produce PHA. Inorganic chemicals are a mixture of essential chemicals and biochemicals required for the fermentation process. Energy consumption comprises stirring during the fermentation process, pumping in and out of the fermentation media from the reactor and maintenance of the fermentation media temperature at about 37 °C. Similarly, water is consumed for fermentation media and downstream processing.

Impact of geographical context:

The comparison of ecological footprint for biodiesel production using electricity mixes from different countries with renewable energy mix for EU, using rape seed oil for biodiesel production (bio_RSO) and diesel production was provided. It turned out that diesel production has the highest footprint with 715,469 m2/t among all these processes, while biodiesel production using the renewable energy mix has the lowest value of footprint 310,771 m2/t.

Task 3.3: Engineering and costing

It was necessary to perform analysis of economic feasibilities of technology to be transferred to the industry for successful implementation. The cost for any product can be divided into three main categories: direct costs, fixed cost and general costs. Direct costs comprise of the expenses which have direct effect on the production rate i.e. raw materials, operating labor and utilities costs. Fixed costs are independent of the production rate and are effective even there would have been interruptions in the operation process. It consists of facility or plant depreciation, taxes and insurances etc. The last but not the least cost section is general expenses which include overheads for maintenance of the plant operations, administrative costs and research and development funds (C.G. Pereira et al. 2007).

Details:

According to data obtained from project partner Argus, the investment cost for microfiltration plant having a capacity of 1 m3/h, considered for acid reclamation, amount to 60,000 EUROS. According to data obtained from a commercial chopper producer company based in Italy, the chopper price varies from 30,000 EUROS to 55,000 EUROS depending on the chopping capacity which varies from 3000 kg/h to 5000 kg/h.

Waste water plant feasibility

In order to accomplish a feasibility study of a waste water treatment plant, it is necessary to calculate the annual water consumption and discharge. In the current project cumulative waste water to be treated is sum of waste water produced from rendering unit, biodiesel production unit, fermentation and downstream processing unit. This calculation is in accordance to the basic assumption of an annual PHA production of 10000 t.

Biogas plant feasibility

According to the available data 4,808,525.36 m3 of biogas is estimated to be produced annually at a production rate of 601 m3/h. Investment costs for the current case is calculated by comparing investment costs for biogas plant having 250 Nm3/h biogas production capacities (BIOGAS Netzeinspeisung 2013). For 601 m3/h biogas production, it is assumed that all the parameters will be calculated using a linear function while a factor of 0.70 is used to calculate fermenter cost.

Infrastructure:

Infrastructure cost for the industrial facility has been considered for an area of about 900 m2 internal floor area. It includes cost break down from feasibility report to turnkey construction facility (Langdom and Wilkes 2006). The calculated infrastructure construction cost is about 896,000 EUROS.

Depreciation:

Life span of the facility is considered 20 years. Using a linear function for all sub processes, the investment cost annual depreciation is calculated.

Operational costs:

Operational cost for the overall process is calculated according to the method described by Haandel and Lubbe (2007). Operational cost for rendering process and biodiesel production process has been obtained from the partner groups. For other sub processes operational cost is calculated according to the described method. The required investment cost is obtained by subtracting rendering and biodiesel production investment cost from the overall investment resulting in 30,253,879 EUROS.

PHA production costs:

PHA production cost is the sum of overall operating costs for all sub processes to produce input materials and chemical cost for 10,000 t PHA production and annual plant depreciation. It counts for 1.68 EUROS/kg of PHA production.

Revenue:

The revenue is calculated using selling prices for the main product PHA and the byproducts, namely high-quality biodiesel and MBM. The selling prices are 4 EUROS/kg PHA, 0.97 EUROS/l biodiesel and 300 EUROS/t MBM.

WP4: characterization, processing of PHA formulations and relevant blends and composites (RDT)

Task 4.1: Preparation and processing of PHA formulations and relevant blends and composites

TERMO in close cooperation with UNIPI carried out processing trials on pilot scale of two different grades of PHA (PHB homopolyester and Poly(3HB-co-4HB) according to lab-scale protocols. The selected materials (PHAs) were scaled-up to kg quantities and submitted to granulation in double screw extruders upon processing of the various powder-formulates in turbomixer. It was demonstrated that, using the available processing equipment, the utilization of Poly(3HB-co-4HB) was beneficial for processing in comparison to PHB.

Task 4.2: Characterization of PHA formulations, relevant blends and composites and processed items

The blends PHB/PS/P(S-MMA) where the PHB was the matrix and the P(S-MMA) the compatibilizing agent showed mechanical and thermal similar to the pristine polymer.

The PS and the PVAc increase the onset degradation temperature of the PHB when are blended with the PHB IN 33% in wt. and 10% in wt, respectively. The blends PHB with PVAc showed good compatibility.

WP5: Ecocompatibility and biocompatibility of the prepared PHAs and relevant processed prototype items (RDT)

Task 5.1: Determination of biodegradability of PHA Formulations and relevant typical prototype items (Blends and Composites) under different environmental conditions as ecocompatibility indicator- biodegradation tests in solid media and in Aqueos Medium Blends and Composites

Task 5.2: Eco-toxicity tests of PHAs and relevant items

PHA formulations and relevant prototypes have been screened for cytotoxicity and genotoxicity assessment by using murine balb 3T3 clone A31 cell line, which is usually considered a sensitive cellular model to verify in vitro toxicity. The possible leaching of toxic compounds from the PHA formulations was evaluated by mean of polymer extracts assay by measuring cell proliferation and Colony Forming Efficiency (CFE), following the ISO-10993/5 guidelines. Carcinogenic potential was evaluated by mean of cell transformation assay (CTA) as indicated in ISO-10993/3. Results highlighted good values of cell proliferation and colony forming efficiency for all the tested formulations and prototypes confirming a complete absence of toxicity. Moreover, no evidence of cell transformation has been detected from the carcinogenic potential evaluation carried out on the tested formulations and prototypes.

Task 5.3: Evaluation of LCA

This task is closely related to the activities in task 3.2. Based on the actual process design, material – and energy balances, an ecological footprint was calculated with the SPIonExcel tool.

The initial footprint for PHA at the beginning of the calculations is about 1,960 m²/kg which is compared to Polyethylene LD (2,500 m²/kg) better in terms of environmental impact. Improvements during the project revealed a footprint for the ANIMPOL-PHA of less than 400 m2/kg PHA, taking into account scenarios of sustainable energy input, and the positive outcomes of the biocompatibility tests.

Task 5.4: Determination of biocompatibility of PHAs and selected relevant prototype items by cytotoxicity and genotoxicity tests

This task is closely related to the activities accomplished in Task 5.2 using the same methodology, but investigating complete PHA-prototype items.

Three-dimensional porous scaffolds based on Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx – HHx=12%) were fabricated by using an in-house (UNIPI) modified additive manufacturing (AM) technique, in the view of exploring the possibility to employ PHAs as polymeric biomaterials for tissue engineering applications. In particular attention was devoted to the preparation of 3D micro-structured scaffolds with defined geometry for bone tissue engineering applications. The manufacturing of scaffolds was performed by means of a ROLAND MDX 40A (Roland MID EURORE, Italy) modified in-house in order to allow for the production of computer controlled 3D scaffolds with the principle of wet-spinning.

Potential impact:

What are the major outcomes of the project?

It has to be underlined that nowadays about 280 Mt of plastics are produced globally with an expected annual increase of about 4%. Thereof, only a minor share of about 5% is attributed to biobased and biodegradable polymers. Nevertheless, currently, we are experiencing a remarkably dynamic biopolymer market with an impressive increase of the volume and range of products: in 2010, the market value reached a magnitude of 10^10 US-dollars with a clearly upwards trend. Only from 2008 to 2015, the global production of biopolymers is estimated to increase from 180 to 1710 k tonnes. Since the launch of ANIMPOL, the price for petrol, the raw material for production of 'classical', highly recalcitrant polymers, has fluctuated up and down. At the same time, the political situation in many petrol-exporting countries like Bahrain, Libya etc. became highly instable, and the future in other important petrol exporting countries is not predictable. In addition, the public awareness and sensibilization for ecological concerns like the greenhouse effect, global warming, was continuously increasing since the start of ANIMPOL, indicated by the strong presence in media and the controversial discussions dealing with these topics. The ANIMPOL consortium is convinced that, based on the elaborated results, a strategy can be presented that at least to a certain extend provides a solution for the discussed concerns.

Who wants the elaborated solutions?

The project will bring together the players of the waste resources from slaughterhouses, rendering industry, and waste fractions of the biodiesel production with polymer producing and/or processing industry. Large companies and SMEs involved in the meat-processing sector from Austria, Germany, Slovenia and Italy have already addressed their interest in the elaborated results and solutions.

Transnational impacts

Already now, transnational partnership between industry and academic institutions in the participating countries is facilitated with balanced benefits between scientific partners and the companies. The project outcomes provide new input to European industrial and scientific institutions and create economic and ecological benefits by converting waste into value added, environmentally benign materials. For synthesis of progress in environment conservation and economic value addition by reduction of pollutants, biotechnological polymer production features a future-oriented solution with a high public acceptance. The created network will be of strategic value as well for the academic institutions as for the companies involved.

Socioeconomic impacts

All industrial partners in the project can profit from the highly qualified scientists and technicians to be educated within the planned cooperation at the research institutions as valuable human resources. Grace to ANIMPOL, jobs was created both at the research institutions and at the industrial partners. A total of 72 jobs were created grace to ANIMPOL, corresponding to about 35 person/years; among those, about 20 people were exclusively recruited for the project. Many of these people maintained their employments at the companies and universities even after the end of the project.

Successful gender balance

Gender balance, as promised at the negotiation process for granting ANIMPOL, was realized. Among the about 20 positions exclusively created for ANIMPOL, more than half were devoted to female co-workers. Although all work package leading positions were occupied by men, 10 women with highest qualification (doctoral degree) were involved in the research.

The consortium was able, by addressing especially female student by the actions of open labs etc., to achieve a share of female PhD students active for ANIMPOL of 56%, and a similar value for students having accomplished their master thesis on ANIMPOL tasks.

What further development steps will be needed?

Now, an up-scaling of the developed processes should be done in a pilot plant. This should be accomplished by interested lead-users of the project results, of course with the help of the academic partners involved. Several interested industrial companies from different European countries already contact the consortium leader. Especially the implementation of ANIMPOL-derived plastic materials for food packaging seems to be the most likely application in the next few years.

After a successful pilot phase, the final step has to be towards the market launch of bio-polymeric products produced by the strategies elaborated in the project.

Dissemination and exploitation

The development of dissemination and exploitation strategies required the highest degree of collaboration between all participants in the consortium in order to achieve a common consensus about the 'conversion' of the results obtained during the research and to make them accessible for the lead companies and users and finally to the broadest possible and sustainable use. Due to the efforts done by all partners, the representation of ANIMPOL in public, the number of scientific publications and the contributions to conferences and fairs by far exceeded the expectations of the Consortium at the beginning of the project. Dissemination of the results as a central item of ANIMPOL was done by a huge number of written and oral publications in research journals, industrial journals and books as well as at diverse conferences. Especially the huge number of national and international conference contributions (oral and poster presentations) presenting the concepts of and data from the ANIMPOL project has to be underlined.

Patentable know-how from ANIMPOL

No option for the ANIMPOL consortium is to manage intellectual property (IP) merely to exclude rivals (and by doing so blocking innovation) or merely to limit the exploitation by piling IPRs for potential cross licensing of single partners or to put technology without an explicit business case consideration to the public domain. The subsequent section provides an overview of accomplished and envisaged patenting activities.

List of websites:

http://www.anipol.tugraz.at

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