Polyhydroxyalkanoates (PHA), the only group of “bioplastics”
“Bioplastics” such as PHA must be competitive with petrochemically manufactured plastics both in terms of material quality and manufacturing economics. Cost-effectiveness calculations clearly show that PHA production costs, in addition to bioreactor equipment and downstream technology, are mainly due to raw material costs. The reason for this is PHA production on an industrial scale currently relying on expensive, nutritionally relevant “1st-generation feedstocks”, such as like glucose, starch or edible oils. As a way out, carbon-rich industrial waste streams (“2nd-generation feedstocks”) can be used that are not in competition with the supply of food; this strategy not only reduces PHA production costs, but can also make a significant contribution to safeguarding food supplies in various disadvantaged parts of the world. This approach increases the economics of PHA production, improves the sustainability of the entire lifecycle of these materials, and makes them unassailable from an ethical perspective.
In this context, our EU-funded projects ANIMPOL and WHEYPOL, carried out by collaborative consortia of academic and industrial partners, successfully developed PHA production processes, which resort to waste streams amply available in Europe. As real 2nd-generation feedstocks”, waste lipids and crude glycerol from animal-processing and biodiesel industry, and surplus whey from dairy and cheese making industry were used in these processes. Cost estimations made by our project partners determine PHA production prices below 3 € (WHEYPOL) and even less than 2 € (ANIMPOL), respectively, per kg; these values already reach the benchmark of economic feasibility.
The presented studies clearly show that the use of selected high-carbon waste streams of (agro)industrial origin contributes significantly to the cost-effectiveness and sustainability of PHA biopolyester production on an industrial scale.
- Animal-processing industry
- polyhydroxyalkanoates (PHA)
- raw materials
- waste streams
Nowadays, we are dealing with plastics in many situations of our all-day life; in most cases, “plastics” are polymers stemming from petro-chemistry. Based on favorable material features such as low density, high resistance, and well-optimized manufacturing processes, plastics are by far the fastest emerging group of materials used for manufacturing of customized items. In this context, plastics are needed for packaging of diverse goods, agriculture, electronics, construction industry, transportation, health care, or the sport and leisure sector. However, based on the limitation of petrochemical resources and the recalcitrance of plastics towards biodegradation, we currently observe growing global concern related to traditional plastics of petrochemical origin. Reliable estimates speak about a pile of 8 to 9 x 109 t of plastics that have been made globally in recent decades (1). In the
face of a current worldwide plastic production of almost 4 x 108 t annually with an enormous uptrend noticed especially in emerging and developing countries, which are characterized by boosting industrialization (2), it becomes clear that the enormous quantities of spent plastics (already surpassing 15 x 107 t annually) urgently require proper disposal (3).
Because of the high recalcitrance of plastics towards (bio)degradation, other methods of disposal are frequently used, often simple landfilling, or they are even just deposited in the environment; this fate struck about 79% of the total amount of plastic ever produced, and resulted in growing piles of plastic waste and plastic waste distribute all over the planet (2). A currently hot topic is the disastrous pollution of the oceans, the origin of all life, by an approximate amount of 2 x 106 t of plastic waste entering marine environments mainly via rivers and coastal sewers; as one of the consequences thereof, “microplastics” endanger the complete food chain (4). Alternatively, estimated 12% of all plastics produced to date were incinerated for energy generation, leading to formation and release of greenhouse relevant and toxic gaseous emissions (1). Alternatively, recycling of spent petrochemical plastics is a generally accepted technology and regarded as “green” and progressive; however, plastic recycling only works to a restricted degree because of the need for a certain purity of spent plastic regarding types of plastics and pollutions, and the material fatigue and quality loss with every recycling cycle (3,5).
As a biological alternative to petrochemical plastics, one can fall back on solutions provided by Mother Nature. In this context, microbially synthesized polyhydroxyalkanoate (PHA) biopolyesters are these days in the focus of microbiologists, systems and synthetic biologists, material scientists, and chemical engineers. This is easily understood due to their beneficial properties such as biodegradability and versatility; these features make them attractive for numerous applications as known for traditional plastics (6,7). PHA were first observed and described almost one century ago by Maurice Lemoigne as light-refractive inclusion bodies in the Gram-positive bacterium
To address the use of inexpensive carbon sources, biorefinery concepts are developed these days, which resort to carbon-rich side streams of diverse (agro)industrial processes to be used as “2nd-generation feedstocks” for PHA production (15,16). In this context, as the first core topic of this review, lactose-rich surplus whey from dairy industry is another viable feedstock for PHA production (17). Further biorefinery concepts are based on PHA production from waste lignocelluloses, such as bagasse, forestry residues, or straw (18), side streams from sugar mills (19), paper industry waste (20), urban sewage (21), nutrient-rich effluents of olive oil production (22), or, in the case of autotrophic PHA production strains, carbon dioxide-rich exhaust gases stemming from power plants (23). Moreover, connecting PHA production to the animal-processing industry was accomplished; here, PHA-producing microbes were supplied with low quality biodiesel obtained by transesterification of high-fat animal waste in addition to crude glycerol and innards, which are additional waste streams from this industrial process, as detailed as the second core topic of this review (24).
In addition to the raw material issue, the downstream processing technology for the efficient and sustainable isolation of biopolyesters from microbial biomass, a crucial step in the PHA production chain, is currently under discussion. In this context, PHA extraction by solvent-based techniques, and various methods for chemical, enzymatic, and mechanical disintegration of the non-PHA (“residual”) biomass are comprehensively summarized in the current literature. However, all these methods have certain disadvantages either from an economic point of view, for ecological considerations, safety aspects, low PHA recovery yield, product pureness, or because of scaling difficulties, which are industrially critical indeed (25, 26, 27). Recent developments to optimize PHA recovery involve the application of harmless solvents (28), supercritical fluids (29), or ionic liquids (30). As a new curiosity which, nevertheless, might have some potential for application, PHA granules of surprising purity can be excreted by some animals like the meal worm
At present, robust microbial PHA producers with well-studied genome and enzymatic machinery, and a broad spectrum of inexpensive substrates utilized by them are in the spotlight of PHA researchers focusing on microbiological and genetic aspects. Especially exploration of extremophilic microbial strains settling in challenging environments, such as halophile microbes isolated from salt lakes, saltworks, or marine waters, is strongly increasing these days. Application of such production strains enables designing simple, flexible and robust cultivation processes, which can be energy-saving due to low or even without any sterility requirements (32, 33, 34, 35).
From the perspective of the chemist, PHAs are polyoxoesters of hydroxyalkanoic acids. To date, a huge number of PHA homo- and heteropolyesters of different molecular composition, mi-crostructure, thermal and physical properties were detected in living microbes. Properties of PHA are fixed by the monomers building up the polyester, and by the polymer´s microstructure, hence, if the PHA are blocky structured or if their monomers are randomly distributed. The types of monomers present in PHA are determined by the substrates provided to the cells, hence, the combination of main carbon source and co-substrates (precursors) of a structure chemically related to given monomers (6,7). The molecular mass of PHA, which can range to some MDa, is strongly influenced by the type of carbon source, the production strain, and the activity of the enzyme PHA synthase, which polymerizes monomers to PHA polyester chains; PHA synthase activity, in turn, is determined by the process conditions (pH-value, temperature, etc.) and the current carbon source availability. To cope with the obvious complexity of PHA molecules occurring at the same time in a biological system in terms of monomer composition and degrees of polymerization, the expression “PHAome” was recently introduced as a relevant technical term in the PHA-related scientific literature (36).
The improvement of large-scale productivity, cost efficiency, and strain performance demands optimization procedures of different process steps. This includes the quest for new wild type and genetically modified microbes, development of a process design adapted to the kinetic characteristics PHA production, and ecologically benign and efficient techniques for separation of PHA and residual non-PHA biomass. Only the synopsis of these steps allows ecologically feasible and sustainable manufacturing of PHA (6,37,38).
Purified sugars, expensive fatty acids, or edible oils are traditionally used as substrates for PHA biosynthesis; therefore, about 50 % of the total PHA production cost is allotted to the raw materials (substrates) (39). As a paradigm shift, inexpensive carbon-rich substrates are currently exploited to enable economically practical PHA production. Especially carbonaceous waste- and byproducts of agriculture, forestry, horticulture, and food industry have already been tested as substrates for PHA biosynthesis on different production scales. Here, it should be noted that the choice of feedstocks can impact PHA quality regarding the polymer composition on the level of monomers, the molecular mass (39, 40, 41), and can affect PHA´s sensory quality in terms of odor or pigmentation (42).
Such inexpensive substrates are expected to meet various basic requirements:
Regarding suitability for storage, easily perishable raw materials such as molasses can be converted via anaerobic fermentation towards storable intermediates like lactic acid or volatile fatty acids, which, in a second process stages, act as feedstocks for aerobic PHA production. This strategy was successfully demonstrated by using green grass juice, which was anaerobically fermented towards ensilage; pressed silage juice, rich in lactic acid, minerals, and nitrogen-containing compounds, turned out to be a feasible, stable feedstock to thrive the PHA producing organism
These restrictions clearly demonstrate that, although the idea of PHA production from inexpensive substrates is generally accepted by the scientific community, above listed obstacles need to be considered when planning a new PHA production process based on “2nd-generation feedstocks” on industrial scale. Moreover, it was previously reported that applying inexpensive substrates reduces PHA productivity when compared to production setups using purified substrates. This is often due:
Insufficient conversion of a new feedstock by microbial production strains can often be overcome by adaptation of organisms to the new substrate by consecutively thriving the strain in a series of pre-cultures containing this substrate. Metabolic bottle necks in substrate conversion can often be solved by tools of genetic engineering. Low carbon source concentration in the substrate feed solution makes the process less efficient, particularly in the case of fed-batch cultivation setups; here, the low concentrated feed solution dilutes the fermentation broth, thus increasing the volume. Low overall productivity in turn encumbers the aspired saving of production costs, because the bioconversion would take longer process time, and a higher total quantity of substrate to generate the same quantity of product. This causes higher operating and capex costs, particularly in large-scale production (44).
Therefore, upstream processing of feedstocks needs optimization to generate feed solutions rich in carbon source and low in inhibiting compounds. Such approaches were demonstrated for the feedstock whey. Because sweet whey contains only about 4-5 wt.-% of the main substrate lactose, an ultrafiltration step can be applied to separate the protein-rich fraction (whey retentate) from the lactose-rich fraction (whey permeate), which contains about 20-21 wt.-% lactose. As shown by Ahn
For removal of inhibiting by-products generated by hydrolysis of inexpensive raw materials such as lignocelluloses like furfural, 5-hydroxymethyl furfural, and others, a simple solution can be to remove such compounds by separation with charcoal or lignite. This approach was only recently performed by Kucera
As major by-product of biodiesel production via alkaline methanolysis of lipids, crude glycerol phase (CGP) is generated at increasing global quantities; CGP contains about 65 wt.-% of the substrate glycerol plus various additional components such as water, biodiesel residues, free fatty acids, acylglycerides, soaps, and minerals. To be used as fermentation substrate, CGP requires demethanolization to remove this methanol, which is strongly inhibiting for many microbes. (52,53). Methanol removal can be managed by vacuum-assisted evaporation, or thermally using so called “preheat tanks” (53, 54, 55). Moreover, the glycerol content in CGP can be increased by removing water via distillation, vacuum dehydration, or more advanced phase separation procedures. In addition, some bacterial specialists are able to accept both methanol and glycerol as carbon sources, as successfully demonstrated by Braunegg
Direct conversion of strongly inhibiting substrates by metabolically versatile organisms was reported by Ward
During cheese and casein production processes, whey is generated at huge quantities as the aqueous side stream of the acidic or enzymatic coagulation of milk casein (“transformation”). Whey represents up to 95% of the entire volume of processed milk (59), with about 9 L of whey being generated per kg of produced cheese (60). Reliable data of OECD and FAO for 2008 estimate a global whey production of about 1.60*108 t per year, with an annual increase by 1-2% (59).
Whey accrues as a surplus stream in different global areas (61), mainly in Europe and North America; in 2008, about 5 x 107 t of whey were produced in the EU only. Because of cow milk being the worldwide leading raw material for cheese production, bovine whey embodies the major fraction of the global whey quantity (62). In addition to countries of the Northern world hemisphere, increasing volumes of whey are currently also generated in southern countries; for example, 270,000 tons annually are generated in India, constituting a real waste stream there without any further use (63).
As a matter of fact, whey has some market applications, such as for production of dietary supplements for body builders, sweets, whey-based drinks, technological additives for processing meat or producing ice cream, chocolate substitute, or baby food; moreover, also the cosmetic and the pharmaceutical sector has a certain demand for whey (63). Although whey, because of its high content in proteins, sugars, lipids, vitamins, and minerals like calcium has a considerable nourishing value with 100 g of whey corresponding to more than 100 kJ of nutritional energy, its application in the food sector is restricted by about 75% of all adults worldwide suffering from lactose intolerance (hypolactasia) caused by insufficient activity of the enzyme β-galactosidase (lactase). Particularly, hypolactasia is frequently diagnosed in South European regions and countries of the Southern world hemisphere (64). Together with the current price drop for whey powder, this makes clear that alternative policies for whey valorization are needed. In parallel, we witness a current controversy on the excessive volume of milk, which is currently flooding the European market. Upgrading whey to an innovative raw material would open the door to support milk-producing companies, and offer stay-options for agrarians in underprivileged regions, which, in turn, could generate socio-economic benefit.
Traditional treatment of whey encompasses dumping into aquatic environments, disposal in caves, dispersion over fields, or application as livestock feed. However, whey´s lion´s share these days is disposed as waste, resulting in tremendous ecological contamination due to its high biochemical and chemical oxygen demand of more than 40,000 ppm and 50,000 ppm, respectively. Due to depletion of dissolved oxygen, whey disposal into the sea causes deterioration of aerobic marine microbes and animals (65). Lactose, whey´s major carbon compound, and the protein fraction are mainly responsible for the high environmental burden caused by whey. Releasing whey on fields to manure them severely affects the physical and chemical structure of soil, which negatively affects crop yields (66). Currently generated quantities of whey by far surmount those to be used as livestock fodder for ruminants and pigs. In Italy, feeding pigs with whey for production of ham of protected regional label (“
During the project WHEYPOL, financed by the 5th Framework programme of the EU, different gram-negative microbial species were tested for production of PHA biopolyesters of different monomeric composition, using either hydrolyzed or non-hydrolyzed whey permeate as carbon source (68). Here, the studied eubacterial production strains
In contrast to Gram-negative microbes, the use of Gram-positive organisms for whey-based PHA biosynthesis is only scarcely described in literature. However, Obruca and colleagues used
Because many promising PHA producing microbes are not able to convert lactose directly due to lacking or deficient β-galactosidase activity, viable hydrolysis techniques are required to split the disaccharide lactose into the monosaccharides glucose and galactose. In analogy to lignocelluloses hydrolysis under harsh conditions, whey permeate hydrolysis using strong acids (typically HCl or H2SO4) at high temperature and pressure generates a brown liquid rich in various inhibiting compounds, which requests further purification. Enzymatic hydrolysis displays a convenient alternative to the use of acids; only one enzyme (β-galactosidase), which operates at mild conditions of temperature and pH-value, is needed to completely convert lactose into the monosaccharides; this is different to the multi-enzyme cocktail requested to hydrolyze lignocellulose materials into a sugar solution. In the WHEYPOL project, inexpensive and customer-friendly β-galactosidase formulations (MaxilactTM, company DSMZ), which are used in food technology for lactose hydrolysis, were successfully applied to prepare the substrate for PHA production (79).
Alcalase was used by Obruca and colleagues to hydrolyze whole whey (WWH). WWH was used as a complex nitrogen- and carbon source to increase cell growth and PHB production by
Whey proteins were also used to coat poly(ethylene) (PE) or poly(ethylene terephthalate) (PET) plastic films; this way, bio-based, recyclable, and biocompatible materials with high oxygen and water vapor barrier were designed, which could potentially be used for food packaging; especially the low oxygen permeability and moisture transfer attracts attention for these new materials for extension of packaged food shelf life (reviewed by (81)). When using a commercial compostable carrier film (Bio-FlexR F 2110TM) instead of PE or PET for coating with whey protein, resulting composites are readily biodegraded and composted without negatively impacting compost quality (82). Further developments in this direction should aim at the substitution of PE or PET by other bioplastic films, most preferably made of PHA produced from whey-permeate.
An estimated annual quantity of 5 x 105 tons of lipid-rich waste is generated by the European animal processing industry, which includes slaughterhouses, rendering companies, or meat-converters (83). The project ANIMPOL, financed by the 7th Framework Program of the EU, consisted of a consortium of seven academic and four industrial partners (84). Based on the experiments carried out in the context of the ANIMPOL project, it could be demonstrated that lipids can expediently be extracted from these waste materials, and undergo transesterification to generate fatty acid methyl esters (FAME), which can be used as biodiesel, a renewable biofuel. In addition to the huge quantities of animal-based waste, the “mad-cow-disease” (BSE) crisis some years ago, which provoked the question of innovative, value-adding and sustainable conversion of meat and bone meal (MBM), gave also reason to realize this project (85). Hence, the project consortium searched for value-adding alternatives to the contemporary incineration of animal-based waste. From a holistic point of view, the results should support the food industry sector, and moreover, also contribute to food security.
Looking at the fatty acid pattern of animal-based lipids, we notice a large share of saturated fatty acids, mainly pelargonic (C9:0), pentadecylic (C15:0), palmitic (C16:0), margaric (C17:0), or stearic acid (C18:0); after transesterification of such lipids, these saturated fatty acids also occur in animal-based FAME (86). However, a high share of saturated fatty acid esters (SFAE) in biodiesel causes increase of the cold filter plugging point; this impedes its use as engine fuel at low temperature by precipitation of solid SFAE particles. Alternatively, the SFAE and unsaturated FAME fraction can be separated by simply precipitating SFAE via cooling; by a subsequent filtration step, SFAE can be easily recovered, and finally converted biotechnologically to PHA biopolyesters by various powerful microbial species. Taking into account a theoretical conversion yield of 0.7 g PHA per g SFAE (based on the experimental data from ANIMPOL) and the available amounts of animal-based waste lipids, 35,000 annual tons of PHA could theoretically be produced (83). The unsaturated FAME fraction remaining after separation of SFAE can be used as valuable “2nd-generation biofuel”.
This transesterification process generates about 0.1 kg CGP as main side product per kg lipids (triacylglycerides) (87). Calculating with the entire biodiesel quantity produced these days in the EU (2—3 x 107 tons per year), more than 2 x 106 tons of CGP are generated as by-product, which is considerably exceeding the quantities of glycerol needed for its current uses in the food or cosmetic sector (83). As mentioned before, various microbial species grow well on glycerol as sole carbon source, and, under challenging conditions, transform this triol into PHA (88). If used to generate microbial biomass, more than 0.4 g biomass or PHA can theoretically be produced per gram of metabolized glycerol (89). Regarding the amounts of waste lipids generated by the European animal processing industry, more than 20,000 tons of PHA-containing biomass are accessible per year only from the accruing CGP (90, 91, 92).
Already two decades ago, Cromwick
Follow-up experiments with
During the ANIMPOL project, animal-based SFAE was used as substrate instead of tallow for bioreactor cultivation of
Follow-up experiments aimed at achieving a higher fraction of 3HV monomers in the copolyester by increasing the amount of those compounds in the substrate, which have an odd number of carbon atoms; higher 3HV fractions were aspired in order to decrease the copolyesters brittleness,
This above described SFAE-based process was repeated by replacing SFAE by glycerol as sole carbon source. Also here, a controlled aerobic fed-batch bioreactor process with
Low structured mathematical models were developed by ANIMPOL consortium members for the two discussed PHA production processes by
In analogy to the experiments presented by Cromwick
Repeating this process with the production strain
After the end of the ANIMPOL project, Riedel and colleagues applied native industrially rendered waste animal lipids without prior conversion to FAME.
Based on the ANIMPOL results from the experiments described above, Kettl
Shahzad and colleagues accomplished a more detailed environmental assessment of PHA manufacturing starting from slaughterhouse waste as raw material, again based on elaborated ANIMPOL data. These authors compared, among other aspects, the influence of using diverse energy sources, which, in turn depends on the usual energy mix applied in different regions; this was used to evaluate the impact of the location of a projected PHA production plant on economic viability and process sustainability. The energy supply aspect is crucial since the rendering process used industrially for lipid recovery from the waste streams displays an exceedingly energy-consuming step; consequently, the energy mix (fossil, renewable, nuclear) prevailing in different global regions massively dictates the ecological footprint of the ANIMPOL-based PHA-production process resorting to animal residues. As a general suggestion, the authors concluded that transport routes for raw materials to the PHA production plant have to be as short as possible in order to achieve the highest possible profitability. As the most decisive result of the study, the authors identified a range of ecological footprint, expressed as surface area needed to produce one t PHA, between 373,000 m2for the production plant being located in Norway (more than 90 % of energy produced by hydropower) and 956,000 m2 for a plant operated in France (high share of nuclear energy in the usual energy mix); these footprint values are significantly lower than previously published results assessing the environmental impact of PE, which amounts to about 2.500,000 m2(92).
Later, Narodoslawsky and colleagues studied different other factors that influence the environmental impact of the ANIMPOL process and other biorefinery-like PHA production processes resorting to inexpensive substrates.
Only recently, a comprehensive economic study for the ANIMPOL biorefinery concept including the utilization of additional fractions of the animal processing was accomplished by Shahzad and colleagues. This study covers the utilization of SFAE (biodiesel of low-quality) as main substrate, and hydrolyzed innards as complex nitrogen source; in this calculation, current market values for MBM (use as fertilizer) and the unsaturated FAME fraction (use as biofuel) are considered, showing that these fractions should not undergo bioconversion, but should rather be sold to create more value for the overall process. The techno-economic analysis presented in this study showed that PHA production cost fluctuates between 1.41 €/kg and 1.64 €/kg depending on whether one considers the innards as a waste stream, or if their market value is considered; for unsaturated FAME fraction (sold as biodiesel) and MBM, fixed costs of 0.97 €/L and 350 €/t, respectively, were used for cost-accounting. The impact of market value fluctuations for biodiesel, MBM, and innards, on the final PHA production price, and the return of investment time were determined. The most important result was that, depending on the current market situation, return of investment time for a plant generating 10,000 annual tons of PHA varies between 3.25 and 4.5 years (24).
The present article demonstrates that the production of PHA biopolyesters is complex, and particular steps in the production chain must be taken into account to make the manufacturing process on an industrial scale both economical and sustainable. The upgrading of a wide variety of high-carbon waste streams from a variety of industries will provide these companies with the opportunity to make sustainable use of their waste streams, while helping to reduce the current environmental impact of these industrial wastes. In addition, food and feed reserves can be saved because nutritionally relevant raw materials that were previously used for biopolyester production no longer have to be used for this purpose. In addition, it should be kept in mind that many of these carbon-rich waste streams occur in those parts of the world where the labor market situation is considerably strained. The integration of biopolyester production into production lines of such companies, where the raw materials (whey, low quality biodiesel, crude glycerol, lignocelluloses, etc.) are directly generated as a waste stream, could have a positive effect on the labor market situation in many eco-socially disadvantaged areas. In order not to contradict the noble pattern of sustainability of the entire biopolyester production process, special attention must be paid not only to the raw material issue, but also to the downstream processing technique. This requires that such sustainable and novel PHA recovery methods, which have already delivered excellent results on a laboratory scale, must finally be scaled up to test their industrial applicability. In summary, the synergistic collaboration of diverse scientific disciplines, as well as the close interaction between academia and industry, is required to successfully manage the long-awaited transfer of lab results to industrial maturity. This transfer must necessarily be based on the principles of “Cleaner Production”, “Zero Emmission”, and “Zero Waste” in order to be able to produce biopolyesters as promising materials in the most sustainable way possible.