Enhancements In The Profitability Of Pha Production

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6.2.1 Factors Influencing the Profitability of Bioplastic Production

Conventional plastics like polyethylene (PE) and polypropylene (PP) are produced at a price of less than US $1/kg. In 1995, the Monsanto Company sold PHA products at about 17 times the price of petrol-based plastics.

It is clear that to achieve a truly cost-effective process, all production steps must be taken into account [15]. Hanggi pointed out that the raw materials constitute the major part of the production cost for biopolymers [16]. Recent studies explicitly show that PHA production from purified substrates such as glucose or sucrose has more or less been optimized [17]. Therefore, it is of importance to concentrate further process development on cheaper carbon sources as basic feedstocks.

Beyond the expenses for raw materials, improving the efficiency of biotechno-logical production in bioreactors (optimization of process parameters such as dissolved oxygen concentration that can strongly influence product yields, for details see Section 6.2.6) is decisive for cost effectiveness.

After biosynthesis of the polyester, the needed recovery process (typically a solid-liquid extraction procedure) can constitute another important cost factor, especially in large-scale production. Here extraction solvents that can easily be recycled will be of interest [18]. To avoid leaving the patterns of sustainability solely in biopolymer production, it will be indispensable to concentrate the development of new extraction processes on recyclable solvents that are also of an environmentally benign nature, such as lactic acid esters [19]. Typical precarious solvents like chloroform will have to be avoided.

Product quality of PHAs is very much dependent on the polyester composition. PHB homopolymer, a bioplastic that is easy to produce from a variety of cheap feedstocks, constitutes a material with restricted possibilities of commercial application due to its brittleness and high glass-transition point. Varying the intramolecular composition of PHAs, polyesters with properties ranging from crystalline thermoplasticity to characteristics of typical elastomers can be produced. This can be achieved by supplementing the cultivation medium with cosubstrates that can act as precursors for co- and terpolyester production. In the polymer chains of these co- and terpolyesters, building blocks such as 3-hydroxyvalerate (3HV) or 4-hydroxybutyrate (4HB) are incorporated. These building blocks possess a number of carbon atoms in their side chains or backbones that are different from 3HB and that disturb the crystalline PHB lattice. In 1987, Byrom discovered that poly-(3HB-co-3HV) could be produced on a large scale by supplementing a fed-batch culture of a glucose-utilizing mutant of Alcaligenes eutrophus (recently known as Wautersia eutropha) with glucose and propionic acid (precursor for 3HV formation) [20]. Lefebvre et al. demonstrated that the dissolved oxygen concentration not only affects the conversion rate of the main carbon source toward PHB, but is also of high importance for the rates and especially yields of the formation of 3HV building blocks from propionic acid (see also Section 6.2.6) [21]. In addition, it has been shown that the utilization of valeric acid instead of propionic acid results in a higher proportion of 3HV units [22].

The use of these often expensive precursors to obtain a higher product quality of co- and terpolyesters translates directly to higher costs for the entire process. A possible backdoor solution is the direct conversion of 3HV-unrelated substrates such as sugars or glycerol toward 3HV by some selected organisms. This rare feature can be found among a few wild-type strains belonging to the genera of Norcardia and Rhodococcus [8, 23-25]. Koller et al. described the formation of poly-(3HB-co-<S-10%-3HV) from whey sugars and crude glycerol phase without the cofeeding of precursors using an osmophilic wild-type organism (see also Sections 6.2.4 and 6.2.5) [26].

It is estimated that, at an annual production scale of 100,000 t of PHB, the production costs of PHA will decrease from US $4.91/kg to US $3.72/kg if hydro-lyzed corn starch (US $0.22/kg) is chosen as carbon source instead of glucose (US $0.5/kg) [15]. But this is still far beyond the cost for conventional polymers, which in 1995 was less than US $1/kg [8]. In 1999, Lee et al. estimated that P(3HB) and medium-chain-length PHA (mcl-PHA) could be produced at a cost of approximately US $2/kg [27], the preconditions being a highly efficient production process and the use of inexpensive carbon sources. Among these substrates, molasses, starch and hydrolyzed starch, whey from the dairy industry (see also Section 6.2.4), surplus glycerol from biodiesel production (see also Section 6.2.3), xylose, and various lipids (see also Section 6.2.2) are available [26, 28-36].

An additional cost factor in normally phosphate-limited production processes for PHAs is the expense for complex nitrogen sources. It was previously found that supplementation of a small amount of complex nitrogen source such as tryptone could enhance 3-PHB production by recombinant Escherichia coli in a defined nutrition medium containing glucose as the sole carbon source [37]. Examples for complex nitrogen sources are fish peptone, meat extract, casamino acids, corn steep liquor, soybean hydrolysate, and cotton seed hydrolysate [33, 38, 40]. Instead of expensive complex nitrogen sources such as yeast extract or casamino acids, cheaper products like silage juice or meat and bone meal can successfully be applied in PHA production processes [26, 39].

6.2.2 Waste Lipids: A Versatile Feedstock

Several waste lipids of different origin can be applied in a variety of sustainable, future-oriented technologies:

Used cooking oil is a waste product that is available in huge amounts [40]. Tallow is an inexpensive source of triacylglyceride (TAG) [41]. From the meat and bone meal (MBM) hydrolysis process that can provide a useful nitrogen source for PHA-producing organisms (see Section 6.2.5), about 11% of lipids remain as surplus material after the degreasing step [42].

In PHA production, biomass has to be degreased before isolation of PHA. Typically 2 to 4% of lipids are isolated from the cells [43].

All of these materials would have to be expensively disposed of unless they can be further used, e.g., in one of the following two ways:

6.2.2.1 Transesterification

Alkaline methanolysis of TAGs results in a mixture of fatty acid methyl esters (FAME) and glycerol. When phosphoric acid is used for the necessary subsequent neutralization, cheap green fertilizers are generated (sodium phosphates). The FAME fraction is mainly used as an ecologically benign fuel (biodiesel) that is gaining increased interest because of its better CO2-emission qualities than diesel from petroleum. For example, the public transit system in Graz (Austria) is expected to complete its switch from petrol to 100% biodiesel fuel by the end of 2005 [44]. The current worldwide annual production is estimated to be 350 million gallons [32].

A smaller share of FAME is converted to fine chemicals such as surfactants [40]. Additionally, biodiesel is directly converted by several bacterial strains toward PHA [45].

6.2.2.2 Direct Utilization of TAGs as a Carbon Source for PHA Production

Tallow is one of the cheapest fats. Production of PHA from tallow has been achieved using Pseudomonas resinovorans. Although the raw material is inexpensive, the process is not profitable due to the low amounts of PHA produced (ca. 15% of cell dry mass) [41].

Pha Production Process

FIGURE 6.2 Different possibilities of utilization of lipids toward higher-value products. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

FIGURE 6.2 Different possibilities of utilization of lipids toward higher-value products. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

The production of poly-(3HB-co-3HV) from olive oils by Aeromonas caviae was described by Doi in 1995. However, the polyester content in cells was rather low (6 to 12%), and therefore this process also was not profitable [46].

Higher amounts of pure PHB (up to 80% of cell dry mass) from different plant oils were produced by Wautersia eutropha [35]. Crude palm oil is a substrate of interests for Erwinia sp. USMI-20. Studies done by Majid et al. show that, using this strain, 46% (PHB in cell mass) was achieved after 48 h of cultivation [36].

Figure 6.2 depicts the different possibilities of utilization of lipids toward production of green plastics (PHAs), green fuels (biodiesel), and other by-products.

6.2.3 PHAs from Raw Glycerol Deriving from Biodiesel Production

Glycerol liquid phase (GLP), the major side stream of biodiesel production from triacylglycerides (see Section 6.2.2), contains about 70% (w/w) glycerol. In all Europe, the total production of biodiesel for 2005 is estimated at 1,925,000 tonnes (t), and the estimate for 2008 is 2,649,000 t, corresponding to 192,500 and 264,900 t of glycerol, respectively [47]. GLP currently constitutes a surplus material. Its utilization leads to an enormous cost advantage compared with commercially available pure glycerol, possessing a market value of 900/t (2002)

10litre Plastic Can Sample

FIGURE 6.3 Scheme of PHA production from the glycerol liquid phase. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

FIGURE 6.3 Scheme of PHA production from the glycerol liquid phase. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

[48]. From the substrate GLP, PHA is produced by different organisms [27, 33]. Figure 6.3 shows the production cycle for GLP from lipid waste and its subsequent utilization as a carbon source in PHA biosynthesis. Starting from crude GLP, and depending on the strain used, a degreasing and a demethanolization step might be needed before the substrate can be applied as a carbon source. The scale-up starting from single colonies of the production strain on solid medium until PHA production on a 300:l scale is demonstrated in Figure 6.3, which also shows the needed downstream processing.

Figure 6.4 depicts the process pattern of production of poly-(3HB-co-3HV) from GLP. Here, GLP was supplied as the sole carbon source without additional precursor feeding. Therefore, an osmophilic wild-type strain was used that converts a broad spectrum of cheap substrates toward PHA production [26]. It is apparent that the organism is accumulating PHA already in parallel with the formation of active biomass (expressed as protein, see Figure 6.4a). Until the end of the cultivation, the cells accumulated PHA up to 70% of their total mass (see Figure 6.4b) at a final copolyester concentration of 16.2 g/l (see Figure 6.4a and Figure 6.4c). During the whole process, 3HV was incorporated into the PHB matrix in a constant amount (8 to 10%). The polymer isolated at the end of the fermentation showed a molecular mass of MW = 253,000 [26].

FIGURE 6.4 Production of poly-(3HB-co-3HV) from the glycerol liquid phase (GLP). Arrows in (a) indicate the refeeding of GLP. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

FIGURE 6.4 Production of poly-(3HB-co-3HV) from the glycerol liquid phase (GLP). Arrows in (a) indicate the refeeding of GLP. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

1 kg PiHB-co-15%HV) can he produced from 3.567 kg lactate

3 567 kg Lactate can be produced from 3.963 kg Lactase

3.963 kg Lactase correspond to 80.878 kg Whey

6 x 1051 Whey can potentially be converted to [7413 l P(HB co- 15%HV)]

5 x lo\ Whey tan potentially be converted to (SISOOO t P(HB-co-15%HV)]

FIGURE 6.5 Possible amounts of PHA from whey in an Italian region and the whole EU from whey by a two-step production process via lactic acid. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

6.2.4 PHA Production from Surplus Whey

Whey is the major by-product from cheese and casein production. Annually, 13,500,000 t of whey containing 620,000 t of lactose constitute a surplus product in the EU [49].

From the feedstock milk, casein is precipitated enzymatically or by acidification. This so-called transformation results in the generation of curd cheese (casein fraction) and full fat whey (liquid fraction). By subsequent skimming, most of the lipids are removed, leaving overskimmed whey. The sweet skimmed whey undergoes a concentration step, where 80% of the water is removed. This whey concentrate is separated via ultrafiltration into a whey permeate (lactose fraction) and a whey retentate (protein fraction with considerable lactose residues). Whereas the retentate

TABLE 6.1

Composition of Different Types of Whey

Lactose Lactic acid Proteins (nitrogen compounds) Lipids

Inorganic compounds

Whey Whey

traces 0.5

Sweet Fermented traces 6-7

Whey Permeate

0.75

Whey Retentate

Source: Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: A Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.

fraction is of interest due to the importance of lactalbumin and lactoferrin for the pharmaceutical industry [50], the whey permeate (containing 81% of the total lactose originally included in the feedstock milk) can be used as a carbon source for biotechnological production of PHAs. Table 6.1 summarizes the composition of sweet whey, fermented whey, whey permeate, and retentate.

Biotechnological production of PHAs from different sugars via condensation of acetyl-CoA units stemming from hexose catabolism is well described, but only a limited number of microorganisms directly convert lactose into PHAs [51]. Depending on the production strain, three possible ways of applying whey for PHA production are viable:

Metabolization of lactose to lactic acid and subsequent conversion to PHA Direct conversion of lactose to PHA

Hydrolysis of lactose to glucose and galactose prior to conversion to PHA

6.2.4.1 Metabolizing Lactose to Lactic Acid and Subsequent Conversion to PHA

The anaerobic conversion of whey lactose by different Lactobacilli strains provides lactic acid with high yields. Lactic acid is a substrate that can be metabolized in a following aerobic fermentation toward PHA by numerous strains, e.g., by Paracoc-cus denitrificans [40]. Figure 6.5 shows the amounts of poly-(3HB-co-3HV) that can be produced by this two-step process from surplus whey of one Italian region and for the whole of Europe (data refer to the entirely produced whey).

6.2.4.2 Direct Conversion of Lactose to PHA

The principal possibility of direct conversion of whey lactose toward PHA using different wild-type bacterial strains is reported in the literature [30, 31, 52, 53]. The cultivation of recombinant E. coli strains harboring PHA synthesis genes for direct utilization of purified lactose has already been studied and is of interest due to high volumetric productivities [54-57]. The application of whey permeate by recombinant E. coli as carbon source for PHA production has also been well investigated [30, 58, 59]. Ahn et al. published the results from a fed-batch fermentation using a cellrecycling system for recombinant E. coli on whey permeate. The determined volumetric productivity for PHB (4.6 g/lh) is the highest reported for PHA production from whey lactose [59].

6.2.4.3 Hydrolysis of Lactose to Glucose and Galactose Prior to Conversion to PHA

Hydrolysis of lactose by p-galactosidase is the first step in biological PHA production from whey lactose (see Figure 6.1). PHA-producing strains exist that show no or insufficient activity of lactose utilization, but they accept the hydrolysis products of lactose, namely glucose and galactose, as substrates [60]. Due to insufficient p-galactosidase activity of the production strain, the hydrolysis rate can be so low that this step is decisive for the total duration of the process. An extension of the process increases costs because of a higher demand of energy and labor. To overcome this problem, lactose can be hydrolyzed chemically or enzymatically prior to the cultivation. This way, glucose and galactose are provided to the microorganisms as carbon substrates [26, 60].

Recently it was shown that the same osmophilic wild-type strain that was used for PHA production from raw glycerol (see Section 6.2.3) is also able to accumulate poly-(3HB-co-3HV) from hydrolyzed whey lactose. Therefore, crude whey was separated from proteins and concentrated directly at a dairy company. The concentrated whey had a lactose concentration of ca. 20% (w/v), which constitutes the upper limit of lactose solubility in water. Lactose included in whey permeate was hydrolyzed enzymatically and used as carbon source for the investigation of poly-hydroxyalkanoate synthesis. Figure 6.6 and Figure 6.7 depict the process pattern of substrate utilization and production of poly-(3HB-co-3HV) from hydrolyzed whey lactose. Also in this case, supplementing of 3HV-precursors was not needed for the

Whey Permeate Powder Bioreactors Lactase Production

50 100

50 100

FIGURE 6.6 PHA production on whey: time curves of substrates (a) and products (b). Arrows indicate the refeeding with hydrolyzed whey permeate. ■ Glucose; X Galactose; ▲ Cell dry mass; X PHA; ♦ Protein.

50 100 120

50 100 120

120

12

b A

^ 100

jTV M

10

a1?

I 80 ei

8

o 60

t

6

(5

J r*

40

A f

4

I

f 1

^ 20

y

2

0

20 40

FIGURE 6.7 Time curves of hydroxyalkanoates (a) and percentages of 3-HV in polymer and ratio PHA/protein (b). (a) ◊ 3-HB; X 3-HV, X PHA; (b) ■ PHA/Protein; ▲ Protein.

generation of 8 to 10% (mol/mol) of 3HV. A final copolyester concentration of 5.5 g/l was obtained until the end of the fermentation. The average molecular weight was 696,000, with a polydispersity index of 2.2 [61].

6.2.5 Hydrolyzed Meat and Bone Meal: a Cheap Nitrogen Source

Severe problems have arisen during the last couple of years in the EU from the emergence of BSE (bovine spongiform encephalopathy). At its peak, the disease had infected 3500 head of cattle weekly in Great Britain [62]. In 2001, this encouraged several scientists at Graz University of Technology (Austria) to research the development of technologies for safe utilization of meat and bone meal (MBM) [63]. One of these novel technologies is the production of alternative cheap nitrogen sources for biotechnological purposes from MBM that was proven to be free of prions. For this purpose, MBM was degreased and further chemically or enzymatically hydrolyzed. Figure 6.8 illustrates the procedure of acidic hydrolysis of MBM and the subsequent neutralization and separation of (predominantly inorganic) precipitates.

The remaining hydrolyzate contains 70% of the original organic nitrogen from MBM (see Figure 6.8) and can therefore be successfully applied as a complex nitrogen source in biotechnological PHA production processes. A fermentation on the 10:l scale using the same osmophilic wild-type organism as described in Sections 6.2.3 and 6.2.4 was reported on hydrolyzed MBM as the sole nitrogen source; crude glycerol from biodiesel production was used as a carbon source. Under these conditions, 1.75 g/l of cell protein and approximately 6 g/l poly-(3HB-co-3HV) were produced [61].

6.2.6 Influence of DOC on the Cost-Effectiveness of PHA Production

Restricted availability of nitrogen, oxygen, or phosphate as initiator for PHA formation was reported several years ago [64]. As a novelty, the effects of a doublelimitation by depletion of nitrogen together with low concentration of dissolved oxygen (DOC) was investigated by Lefebvre et al. in the late 1990s using the strain Wautersia eutropha [21]. In this study, glucose and propionic acid were cosupple-mented in the PHA production phase to enable the biosynthesis of poly-(3HB-co-

MBM 28.5g Organic Nitrogen 10.4% Ash content 29.6%

MBM 28.5g Organic Nitrogen 10.4% Ash content 29.6%

Neutralization Neutralization

Separation

1.25 LMBM hydrolyzed Organic Nitrogen 2.3 g/L

Neutralization Neutralization

Fermentation

1.25 LMBM hydrolyzed Organic Nitrogen 2.3 g/L

Fermentation

Separation

Separation Product

Solids 4.8 g Organic Nitrogen 1.6% Ash content 71.8%

Solids 36.8 g Organic Nitrogen 1.6% Ash content 86.9%

FIGURE 6.8 Hydrolyzed meat and bone meal: production steps and application for fermentation purposes. (From Braunegg, G. et al., Production of Plastics from Waste Derived from Agrofood Industry, paper presented at International Conference Renewable Resources and Renewable Energy: a Global Challenge, Trieste, Italy, 10-12 June 2004. With permission.)

3HV). In direct comparison to a control fermentation with sufficient oxygen supply (50 to 70% of air saturation during accumulation phase), low-DOC experiments (carried out at a DOC between 1 and 4% of air saturation) resulted in a slower production rate for 3HB, probably due to lower glucose uptake rates. On the other hand, the production rate for 3HV increased significantly, although the same enzymes are involved in 3HB and 3HV synthesis. Prior to the experiments, it had to be expected that the slowdown in enzyme activities would also negatively influence 3HV formation! Additionally, it turned out that higher yields of 3HV from propionate are achieved at low DOC.

The described findings can be explained as follows: propionate is typically converted to propionyl-CoA, which undergoes a further condensation with acetyl-CoA to form C5-building blocks. By releasing CO2, propionyl-CoA can easily lose its carbonyl atom, thus building acetyl-CoA [65]. The condensation of two acetyl-CoA units generates 3HB. The experiments done by Lefebvre et al. [21] indicate that this unwanted oxidative loss of CO2 from propionyl-CoA can obviously be avoided by restricting the oxygen supply, resulting in higher yields of 3HV from propionate at the expense of 3HB and, due to the lower glucose uptake, the total copolyester formation. On an industrial scale, one would have to decide, as the case arises, whether the increased 3HV yield from propionate, together with the minimized need for aeration, can economically compensate the lower overall PHA production. This will very much be dependent on the desired end composition of the copolyester. Table 6.2 collects the detailed data for the discussed findings.

It is known that, due to the lack of depolymerase, recombinant E. coli does not require nutrient limitation for PHA synthesis and produces PHA during growth [66]. Because it was noticed that PHA production was increased in recombinant E. coli

TABLE 6.2

Influence of DOC on Production of Poly-(3HB-co-3HV)

Experiment

Mol% 3HV in PHA a

21.5

31.5

PHA in

3HV/Propionate

0.25

0.73

0.44

0.36

0.33

0.23

0.07

0.14

a Data from end of cultivation.

b A total of 16.0 gl-1 of sodium propionate was added; all of it was used by the cells. c A total of 5.65 gl-1 of sodium propionate was added; 4.7 gl-1 was consumed by the cells.

Source: Lefebvre, G., Rocher, M., and Braunegg, G., Appl. Environ. Microbiol., 63, 827-833, 1997. With permission.

when biomass production was gradually retarded, Kim [30] compared PHA production from whey lactose at sufficient and insufficient oxygen supply by changing the maximum agitation speed during different fed-batch fermentations in stirred-tank fermentors. It turned out that cell concentration as well as PHB homopoly ester productivity and total concentration of PHB (g/l reactor volume) increased with higher agitation speed (better oxygen input), whereas the PHB content in cells was highest at an agitation speed of 500 rpm (80% PHB in cells compared with approximately 60% at 700, 900, and 1300 rpm). A further lowering of the agitation speed (300 rpm) did not further positively influence the PHB content (70% PHB in cells) [30]. The increased content of PHA at 500 rpm is most probably due to the reduced formation of non-PHA biomass in combination with a specific PHA production that was not influenced; the decrease in PHA content at 300 rpm might again be due to a restricted specific sugar uptake rate of the cells (see previously described experiments of Lefebvre et al. [21]). In addition to the previously described findings for Wautersia eutropha, the results achieved by Kim [30] clearly indicate the importance of optimizing the oxygen supply during PHA synthesis.

6.3 CONCLUSION

The presented studies clearly demonstrate that a variety of surplus materials deriving from the agrofood industry are of interest for the production of biopolymers. Some of these inexpensive substrates act directly as a carbon source for industrial production of future-oriented bioplastics such as poly-(3HB-co-3HV). To illustrate this point, waste materials like whey lactose from cheese production, crude glycerol liquid phase from biodiesel production, and several waste lipids are discussed. Meat and bone meal are presented as a suitable raw material for complex nitrogen sources that are needed to achieve sufficient concentrations of PHA-producing biomass, a precondition for economical, profitable biopolyester production. The application of surplus materials enhances the cost efficiency of PHA production while reducing the amounts of waste originating from these surplus materials.

The costs derived from the addition of precursors for copolyester production can be mitigated by using microbial strains that convert cheap materials directly into poly-(3HB-co-3HV). Production of these precursors can also be enhanced using new fermentation strategies based on limiting the oxygen supply.

The results obtained look promising in regard to future production of economically and ecologically profitable high-quality bioplastics.

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7 On the Environmental Performance of Biobased Energy, Fuels, and Materials: A Comparative Analysis of Life-Cycle Assessment Studies

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