Process optimization for efficient biomediated PHA production from animal-based waste streams
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Conventional polymers are made of crude oil components through chemical polymerization. The aim of the project ANIMPOL is to produce biopolymers by converting lipids into polyhydroxyalkanoates (PHA) in a novel process scheme to reduce dependence on crude oil and decrease greenhouse gas emissions. PHA constitutes a group of biobased and biodegradable polyesters that may substitute fossil-based polymers in a wide range of applications. Waste streams from slaughtering cattle are used as substrate material. Lipids from rendering are used in this process scheme for biodiesel production. Slaughtering waste streams may also be hydrolyzed to achieve higher lipid yield. Biodiesel then is separated into a high- and low-quality fraction. High-quality biodiesel meets requirements for sale as fuel and low quality is used for PHA production as carbon source. Selected offal material is used for acid hydrolysis and serves as a source of organic nitrogen as well as carbon source for PHA-free biomass with high production rate in fermentation process. Nitrogen is a limiting factor to control PHA production during the fermentation process. It is available for bacterial growth from hydrolyzed waste streams as well as added separately as NH4OH solution. Selected microbial strains are used to produce PHA from this substrate. The focus of the paper is about an overview of the whole process with the main focus on hydrolysis, to look for the possibility of using offal hydrolysis as an organic nitrogen substitute. The process design is optimized by minimizing waste streams and energy losses through cleaner production. Ecological evaluation of the process design will be done through footprint calculation according to Sustainable Process Index methodology.
KeywordsPHA Biopolymers Hydrolysis Animal residues Sustainable Process Index
General: the exigency for novel technologies in polymer production
The implementation of living organisms for production of chemical biopolymers like polyhydroxyalkanoates (PHAs) on an industrial scale constitutes part of “White Biotechnology”, characterized by the utilization of renewable resources as feedstocks and the embedding of the production processes into closed material cycles. The use of renewable resources as an alternative to fossil feedstocks becomes interesting for the chemical sector against the backdrop of rising oil prices underlined by the current development of the crude oil price that amounted to more than 100 USD/barrel (for Brent Crude Oil) in the summer of 2011, which is more than double the price of January 2009, when <50 USD/barrel had to be paid (OPEC 2009). Political developments in several petrol exporting countries, as well as the approaching production maximum of crude oil production, add to market uncertainty, especially for the highly petrol-dependent polymer industry.
Besides increasing market uncertainty, environmental considerations and in particular reduction of greenhouse gas emissions have to be taken into account for any new process providing commodity products. Although processes based on renewable resources, especially when using waste streams from other industries, have a clear advantage in this respect, process development has to take necessary steps to guarantee sustainability of production. Using tools like life cycle assessment (LCA) and cleaner production methods, the reduction of environmental impact for production of polymeric materials, therefore, has to be part of any new process development (Sudesh and Iwata 2008).
PHA biopolyesters and economic challenges in their production
Chemically, PHAs are polyoxoesters of hydroxyalkanoic acids (HAs). In nature, PHA accumulation occurs by a broad range of Gram-positive and Gram-negative eubacterial species and in several representatives of the domain of the Archaea from renewable resources like carbohydrates, lipids, alcohols or organic acids; this accumulation classically occurs under unfavorable growth conditions due to imbalanced nutrient supply. For PHA harboring microbial cells, these inclusions mainly serve as reserve materials for carbon and energy, providing them an advantage for survival under starvation conditions, and enhance the cell’s endurance under environmental stress factors. Under conditions of starvation PHAs are catabolized again by the cells (Chen 2010; Koller et al. 2011).
PHAs attract more and more interest due to the fact that they feature material properties similar to petrochemical thermoplastics and/or elastomers. In contrast to petrochemical plastics, PHAs combine the characteristics “biobased”, “biodegradable”, “compostable” and “biocompatible”; hence, they can be classified as real “green polymers”. If items made of PHAs are composted, they are completely degraded to water and CO2 as the final oxidation products. Here, it has to be emphasized that these final oxidation products are the starting materials for the photosynthetic re-generation of carbohydrates by green plants. This demonstrates that, in contrast to petrol-based plastics, PHAs are perfectly embedded into nature’s closed cycle of carbon, underlining their suitability for replacing polymeric materials based on fossil feedstocks needed for the production of marketable plastic items (Koller et al. 2010).
In order to make biobased and biodegradable polymers like PHAs economically more competitive with common resistant plastics from fossil resources, their production costs have to be reduced significantly. Most of all, the selection of suitable renewable resources as carbon feedstock for PHA production is the major cost-decisive factor in the entire PHA production chain, amounting up to 50% of the entire production costs (Choi and Lee 1999). A viable solution is identified, in the utilization of waste and surplus materials upgraded to the role of feedstocks for the biomediated polymer production. Such materials are mainly produced in agriculture and such industrial branches that are closely related to agriculture (Braunegg et al. 1998; Khanna and Srivastava 2005 Koller et al. 2005a Solaiman et al. 2006; Khardenavis et al. 2007).
Objectives and strategies of the ANIMPOL project
The ANIMPOL project, financed by the European Commission within the 7th framework programme (FP7), aims at the sustainable and value-added conversion of waste from slaughterhouses, rendering industry and waste fractions of biodiesel production. Lipids from slaughterhouse waste are converted to fatty acid esters (FAEs, biodiesel). FAEs consisting of saturated fatty acids generally constitute a fuel that has an elevated cold filter plugging point (CFPP), which can be somewhat limiting in blends that exceed 20% (v/v) FAEs. In the ANIMPOL project, these saturated fractions are biotechnologically converted toward high value-added biopolymers. As a by-product of the transesterification of lipids to FAEs, crude glycerol phase (CGP) accrues in high quantities. CGP is also available as carbon source for the production of catalytically active biomass and the production of low molecular mass biopolymers. This brings together waste producers from the animal processing industry with meat and bone meal (MBM) producers (rendering industry), the biofuel industry and polymer producing industry, resulting in value creation for all players.
According to personal communication with the project partner, the entire amounts of animal lipids from the slaughtering process can be quantified with more than 500,000 ton/year (y). This lipid content is potential raw material for 490,000 ton/year of biodiesel production, containing about 55% saturated biodiesel fraction. This saturated fraction is potential substrate for PHA production. From the saturated biodiesel fraction, the amount of PHA biopolyesters can theoretically be calculated with a conversion yield of 0.7 g/g (Choi and Lee 1999). The annual CGP production in 2008 from biodiesel production has been a reported 700,000 tons (Stelmachowski 2011). If this glycerol is applied for the production of catalytically active biomass, about 0.4 g biomass per gram of glycerol may be obtained.
The ANIMPOL project develops an integrated process, comprising the scientific fields of microbiology, genetic engineering, biotechnology, fermentation technology, chemistry, chemical engineering, polymer chemistry and processing, LCA and cleaner production studies, combined with feasibility studies for the marketing of the final products. This is done in close cooperation of academic and industrial partners. The project aims at solving local waste problems which affect the entire European Union; the solutions are meant to be applied to the entire EU.
Process design development
From the slaughterhouse waste to the PHA, several sub-processes were analyzed. Every decision in the process design is influenced by the fundamental principle of creating an ecological and economic efficient process to use the residual streams for the production of PHA.
In the current process design rendering, hydrolysis, biodiesel and PHA production are key parts. A closer look at the flow sheet of the process design reveals that slaughtering of the animals produces three main streams: meat, non-rendering material and rendering material. Meat is directly sold to the market. Non-rendering material contains manure, digestive tract content, milk and colostrum, etc. while rendering material contains mainly fat, bones and blood.
According to the Regulation No 1774/2002 from the European Union (European Union 2002) rendering material is categorized as risk and non-risk material.
Risk material comprises all body parts, hides and skins from transmissible spongiform encephalopathy (TSE)-suspected and TSE-confirmed animals, pets, animals from zoo and wild animals suspected of being infected with communicable disease. Rendering products obtained by this material can only be used for the production of heat. Tallow is used as direct combustion fuel and MBM is incinerated in an approved incineration plant.
Non-risk material contains all body parts, offal, blood, hides, skin, feather, wool, horns and fur from animals neither having TSE nor suspected of being infected by it. A portion of the non-risk material (selected offal) is used for hydrolysis to produce necessary organic nitrogen source. The rest of the non-risk rendering material is processed to rendering products.
In the rendering process, animal by-products are treated at 133°C and 3 bar for at least 20 min to obtain MBM and also tallow extract. There are several rendering products such as MBM, tallow, blood flour and feather flour, and their classification is based on the input material.
For the process design presented here, a rendering process with the output of 21% tallow, 24% MBM and 55% water is taken into account (Niederl and Narodoslawsky 2004). The tallow is utilized to produce biodiesel and the MBM is sold.
The biodiesel process has an already well-developed design. For the process design in this project, the variation of the feedstock to tallow was considered. According to data from an existing industrial facility producing biodiesel using tallow as feedstock, biodiesel production yields are 96–98%. Biodiesel is tallow methyl ester (TME) produced from tallow provided by a rendering process, through a transesterification reaction with methanol using KOH as catalyst. Following Cunha et al. (2009), 1 kg biodiesel and 100 g of glycerol are produced from 1 kg of tallow using 1:6 molar ratio of methanol to tallow.
TME contains a mixture of saturated and unsaturated fatty acids. The content of unsaturated fatty acids defines the quality of the biodiesel, which is measurable with the cold filter plugging point (CFFP). Analysis shows that the representative TME contains 45% of high-quality biodiesel fraction. Low-quality biodiesel, which contains a high amount of saturated fatty acids, is separated using a crystallization step. The low-quality biodiesel fraction is used as a carbon source in the PHA production, while the high-quality fraction is sold directly.
Acid hydrolysis of offal provides a complex nitrogen source for the fermentation process instead of (more costly) casamino acids.
On an industrial scale, PHA production occurs under controlled conditions in bioreactors, enabling the maintenance of constant process parameters (pH value, temperature, dissolved oxygen concentration) and the operation under monoseptic conditions.
Normally, the PHA production process encompasses two easily distinguishable phases: first, a desired concentration of catalytically active biomass is produced under balanced growth conditions by providing all substrates required by the microbes for unrestricted growth. In this phase, the production of PHA is insignificant compared to biomass formation. In a second phase, the supply of an essential nutrient such as nitrogen, phosphate or minor components is restricted, causing nutritional stress conditions for the microbes. This provokes the redirection of the carbon flux from biomass production toward predominant PHA accumulation (Koller et al. 2008, 2010).
Different operation modes are known for biotechnological PHA production; among them, fed-batch strategies are most widely used on pilot- and industrial scale (Nonato et al. 2001). Here, all substrates are re-fed to the system according to their consumption by the production strain. In this case, cell harvest occurs only at the end of the fermentation batch after pasteurizing the cells in situ in the bioreactor. Fed-batch processes for PHA production are generally stable and highly reproducible as soon as reliable fermentation protocols for the production processes are available. In contrast, the continuous mode is the one that should enable high productivities and constant product quality (Zinn et al. 2003; Sun et al. 2007). The concentration of active biomass, PHA and of all substrates is kept constant as soon as steady-state conditions are reached; under these conditions, cell harvest also occurs continuously. Although not yet widely applied in biotechnological industrial praxis because of a higher complexity of the technical set-up and a higher risk for microbial contamination, continuous fermentation strategies are considered to have a huge potential, also for PHA production. In addition, multistage systems provide different cultivation conditions in each stage and thereby approximate the characteristics of a continuous plug flow tubular reactors (CPFR). It is described that a cascade with at least five reactors in series can be used as a process-engineering substitute for a CPFR (Moser 1988; Braunegg et al. 1995). Most recently, the highly efficient production of PHA using a five-stage continuous bioreactor cascade was successfully demonstrated (Atlić et al. 2011).
Hydrolysis and PHA productivity scenarios
For the optimization of the hydrolysis process, three different scenarios based on PHA productivity were considered. The aim was to figure out the hydrolysate demand and the effects on the process design (i.e., usable waste streams).
It is assumed that the annual PHA production target will be 10,000 tons. According to an optimal fermentation time of 48 h, this leads to 150 batches with 67 ton per batch.
Scenario I is based on average values from current laboratory experiments. This scenario forms the baseline for comparison.
Scenario II is based on optimal fermentation conditions, assuming that the produced cell dry mass1 (CDM) contains 80% PHA and 20% residual biomass. Scenario III is based on the results of other projects using different bacterial strains and feedstock (Nonato et al. 2001). This scenario represents the upper bound for possible improvement of the process optimization within the ongoing project.
PHA productivity parameters referring to fermentation media (FM)
(kg dm/m3 FM)
(kg dm/m3 FM)
(kg dm/m3 FM)
(kg dm/m3 FM h)
In Scenario I, 45.6 kg/m3 CDM is produced containing 30.2 kg PHA (dm) and 15.4 kg residual biomass (dm). Only 4.56%w CDM is produced and PHA productivity is 0.63 kg/m3h PHA, assuming a fermentation time of 48 h.
The biomass matter remains constant in Scenario II; however, the PHA content increases to 62.8 kg/m3 FM. The PHA productivity increases accordingly to 1.63 kg/m3 FM h−1.
Scenario III shows nearly double the PHA output, namely 114.5 kg/m3 FM. The CDM content and PHA productivity increase with the PHA productivity, reaching 2.4 kg/m3 FM h−1.
For a rough estimate of the fermenter size, the best case scenario with 114.5 kg of PHA per m3 FM was used. This leads to a fermenter size of 582 m3. This size was used to calculate the required amount of hydrolysate for the fermentation.
Chemical composition of offal
Derived from this data, the required dry mass from hydrolyzation per batch is 2.9 tons, which will result in an annual consumption of 437 tons of offal dry mass. The average dry mass content of offal is 25.3% leading to an annual demand of offal fresh material of 1,727 tons (Neto 2006).
Offal offer based on 130,000 ton/year rendering plant size and offal demand for the hydrolysis
Weight per animal equivalent (kg)a
Weight per animal animal equivalent wt. (%)
Available material wt. (t/y)
Demand for hydrolysis (t/y)
Different fractions of usable (meat, tradable offal, etc.) and waste (stomach content, condemned material) are assumed according to (Riedl 2003).
Hydrolyzation of the residual material is carried out with 6 M HCl at 120°C using a concentration of 100 kg/m3 of offal dry mass for 6 h (Neto 2006), followed by neutralization using NaOH. Assuming 150 batches per year and necessary offal dry mass of 437 ton/year, leads to 4,370 m3 of 6 M HCl.
Equal moles of base will be required to neutralize the solution because the acid concentration remains constant after the hydrolysis leading to an annual demand of 1,330 tons solid NaOH in the neutralization step, which generates 1,556 tons of the neutralization product NaCl. In the FM, the NaCl concentration is limited with 5 g/l, which is equivalent to 0.07 m3 of hydrolysate.
Process design evaluation
Carbon and nitrogen balance
The carbon and nitrogen are liked to each other in a specific ratio, which has been explained in the following description.
Considering theoretical values for conversion rates (Y) of substrate to biomass or PHA in the fermentation step, the input of carbon source into the system boundary to be finally converted by the production strain in the bioreactor can be roughly balanced. Theoretical conversion rate values are given as: biodiesel: Y = 0.6; glycerol: Y = 0.48; carbohydrates: Y = 0.48; fat: Y = 0.6; and N-substance (considered as carbon source): Y = 0.48 (Choi and Lee 1999; Koller et al. 2005a, 2012).
Carbon balance according to the flow sheet
Biomass yield (%)
PHA yield (%)
Biodiesel (low quality)
Total biomass and PHA
Chemical composition of offal in ANIMPOL
Dry mass (t/y)
“1 kg of nitrogen theoretically corresponds to 7.14 kg of PHA-free biomass providing 28.56 kg of PHA considering a PHA content of 80% in the entire cell biomass”. According to this assumption, the available organic nitrogen is sufficient for 1,243 tons of PHA production.
This process will produce 13,024 tons of biomass containing 10,419 tons of PHA. In the fermentation process, nitrogen acts as the growth limiting factor provoking PHA production. According to our experimental evidence, the ratio between organic nitrogen and inorganic nitrogen is fixed. The available 44 tons of organic nitrogen is sufficient to produce 311 tons of PHA-free biomass. The rest of the PHA-free biomass, which is 2,294 tons, requires 321 tons of nitrogen. This required amount of nitrogen is provided by inorganic source of nitrogen, i.e., NH4OH. It is used to control the reaction conditions as 25% NH4OH (wt/wt) solution. The calculated 25% (wt/wt) NH4OH consumption is therefore 3,213 ton/year, containing 321 tons of nitrogen.
Based on the mass and energy flows from the process design, a first ecological evaluation was carried out Instead of calculating the footprint after the process design is completed, ecological evaluation is used as decision criterion during process development. This provides the possibility of focusing on those parts of the process design that are ecological hot spots. In this process scheme, particular interest will be given on the contribution of the hydrolysis step to the ecological pressure, as this step is distinctively new in the technology concept. The evaluation will be carried out with the Sustainable Process Index (SPI) to cover a broad range of ecological impacts.
Sustainable Process Index
SPI is a life cycle impact assessment (LCIA) methodology which offers the possibility of calculating the ecological footprint for processes (Narodoslawsky and Krotscheck 1995) and has been used for many different applications (e.g., Gwehenberger and Narodoslawsky 2008). For footprint calculation, the freeware program SPIonExcel (Sandholzer and Narodoslawsky 2007) was used.
This methodology can be applied for any good and services (e.g., Kettl et al. 2011)
SPI evaluation of PHA production
Life cycle inventory (LCI) data for 1 kg of PHA
Biodiesel (low quality)
Net electricity EU27
Nitrogen production costs per year
Annual costs (€/year)
Total nitrogen production cost
Total nitrogen production
Total nitrogen production cost per ton
Comparison of nitrogen of annual production costs using HCl reclamation and H2SO4 for hydrolysis
Hydrolysis with HCl
Nitrogen production costs (€/ton)
Hydrolysis with H2SO4
N production (€/ton)
The main part of the footprint for the PHA production derives from the usage of biodiesel (low quality) as carbon source. This is due to the fact that biodiesel is produced from fat by an energy-intensive rendering process. Another main impact to the ecological assessment is displayed by the hydrolysis of the offal material, which uses a high amount of acidic catalyst. The reduction and/or recovery of the required catalyst is therefore of major importance and has to be focused in further process development. The same holds for the biodiesel production, where the footprint reduction potential is high if heat integration is considered. This reduction would directly and effectively influence the footprint for the whole PHA production process.
Economic analysis for hydrolysis
Besides the ecological evaluation, an economical calculation is mandatory to bring the project from laboratory to industrial scale. Especially, investment and operating costs have to be estimated to get an idea about the feasibility for PHA against conventional plastic production, but also for every key part in the process design.
At this stage of development, priority has to be laid on the evaluation of the hydrolysis process to decide if this will be a feasible part of the process concept. Ecological considerations already point out the importance of acid recovery in this step. The nitrogen production costs are compared to the price of inorganic nitrogen available from the market. Prices are obtained from Pitt M, n.d. for NaOH, ICIS, n.d. for HCl and European Energy Portal, n.d. for electricity. The Table 7 represents the production costs for organic nitrogen via hydrolysis.
It can be said that nitrogen obtained from the organic source (offal) is quite expensive as compared to inorganic source of nitrogen (NH4OH), which costs 500 €/ton compared to 12,693 €/ton of nitrogen. It is therefore clear that offal cannot be used as the sole nitrogen source in the process. Hydrolysis, however, provides a high-quality, complex nitrogen source for fermentation, which would otherwise be supplied by high-cost substances like casamino acid and grass silage juice, which cost 928,989.64 €/ton nitrogen and 720,505.49 €/ton nitrogen, respectively, and thus considerably more than nitrogen from offal.
Further optimization scenarios have been taken into account to improve the cost-effectiveness of offal hydrolysis. Different HCl reclamations will lower the production costs considerably. Beside that, a possible alternative hydrolysis agent (H2SO4) has been taken into account. Prices for H2SO4 and Ca(OH)2 have been obtained from Pitt M, n.d. Table 8 shows the annual nitrogen costs using HCl reclamation and as alternative, H2SO4.
The bandwidth for the costs of hydrolysis is hugely dependent on the rate of reclamation, but remain much higher compared to inorganic nitrogen, while the advantage compared to other complex nitrogen sources becomes even more pronounced. Offal hydrolysis is therefore a sensible strategy to lower overall production costs; however, acid reclamation in this process step is a condition sine qua non from ecological as well as economical points of view.
The paper presented a process concept to generate PHA and biodiesel from waste flows resulting from slaughter houses and rendering of animal residuals. Using selected offal via hydrolysis as a complex nitrogen source, as well as glycerol and low-grade biodiesel as a carbon source, is an innovative feature of this integrated scheme of utilizing waste from meat production.
Economic evaluation reveals that the pathway of offal utilization provides a complex nitrogen source that is considerably more costly than mineral nitrogen sources, but is however cheaper than comparable other complex nitrogen sources. The use of this material is therefore limited to providing the necessary complex nitrogen sources for fermentation. The use of inorganic nitrogen is still indispensable due to the microbial requirements. Considering the positive effect of hydrolysate on microbial cultivation during balanced growth, it may only be replaced by other agricultural sources (e.g., silage juice) to shorten the lag time (Koller et al. 2005b), but at considerably higher costs.
Ecological evaluation showed two particular sub-processes to be crucial with regard to the overall ecologic performance of PHA and biodiesel production: the hydrolysis step of offal and the rendering process providing lipids for the biodiesel production. Focus for further process development, besides increasing the PHA yield, will therefore be laid on acid reclamation in the hydrolysis process and heat integration in the rendering step.
The overall process performance at this stage of development clearly indicates the potential of this concept. Using waste material from meat production to provide biodegradable, versatile plastics as well as high-quality biofuel will serve the goal of reducing the ecological footprint of society in general and in particular the reduction of greenhouse gas emissions at competitive costs.
Total biomass (dry matter (dm)) produced in the fermentation process (PHA + residual biomass).
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