White Biotechnology for Biopolymers

CHAPTER 16

White Biotechnology for Biopolymers Hydroxyalkanoates and Polyhydroxyalkanoates: Production and Applications Guo-Qiang Chen, Juanyu Zhang, Ying Wang School of Life Sciences, Tsinghua University, Beijing, China

Contents 1. Introduction 555 2. Strains for Production of PHA 559 3. PHA Produced in Industrial Scale 560 3.1 Fed-Batch Process 561 3.2 Continuous Process 563 3.3 Mixed Cultures 563 3.4 Applications of PHA 565 3.5 Environmentally Friendly Bioplastics for Packaging Purposes 566 3.6 Biofuels 568 3.7 Medical Implants 569 3.8 Monomers as Chiral Intermediates 570 3.9 Smart Materials 570 3.10 Challenges for R&D 570 3.11 Future Prospective 571 References572

1. INTRODUCTION Polyhydroxyalkanoates (PHA) is a family of structurally diverse biopolyesters accumulated by many bacteria as carbon and energy source (Figure 16.1).1 PHA have been exploited with a series of applications including environmentally friendly biodegradable plastics for packaging purposes, biofuels, medical implants, and recently, smart materials.2 PHA monomers are also produced as chiral intermediates for medical or fine chemical applications.3,4 Due to the commercial interests of PHA, global efforts have been made to study these polymers related to synthesis mechanisms, monomer diversity, physiological roles, and controllable production.2 Significant knowledge on PHA has been accumulated, allowing further exploitation of PHA commercial production.2,5,6 Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00018-5

Copyright © 2015 Elsevier B.V. All rights reserved.

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Figure 16.1  Polyhydroxyalkanoates (PHA) is a family of structurally diverse biopolyesters accumulated by many bacteria as inclusion bodies of carbon and energy source.

Based on the monomer structures, PHA have been classified into short-chain length (SCL) ones consisting of monomers of 3–5 carbon-chain lengths (C3–C5), mediumchain length (MCL) ones containing C6–C14 monomers, and random copolymers of SCL and MCL monomers.7 Based on the microstructures, PHA can also be named as homopolymers, random copolymers, block copolymers, and graft polymers (Figure 16.2).8 The combined diversities in both monomer structures and microstructures lead to unlimited possibility to manipulate PHA structures and properties. However, it was found that PHA synthesis is related to many metabolic activities in the microbial cells, resulting into uncontrollable PHA monomer supplies, thus uncontrollable PHA molecular structures, leading to inconsistent properties.9 Through many years of research, it becomes possible to design a PHA structure and achieve designed PHA synthesis by metabolic engineering approaches or recently, synthetic biology approaches.10–12 It is also possible to introduce functional groups into the PHA site chains, allowing further polymer structure modification by chemical grafting.13 The grafting of PHA with functional groups can generate smart materials with pH, temperature, humidity, or even stress responsive behaviors. Typical SCL PHA include poly(lactide) (PLA), poly(3-hydroxybutyrate) (P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (P3HV) and their random or block copolymers such as P3HB4HB, PHBV, PLAHB, etc. Typical MCL PHA are homopolymers of 3-hydroxyhexanoate (PHHx), 3-hydroxyheptanoate (PHHp), 3-hydroxyoctanoate (PHO), 3-hydroxynonanoate (PHN), 3-hydroxydecanoate (PHD),

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Figure 16.2  Based on the microstructures, polyhydroxyalkanoates (PHA) can also be named as homopolymers, random copolymers, and block copolymers.

or 3-hydroxydodecanoate (PHDD) (Figure 16.1). More frequently, bacteria will synthesize random copolymers of these MCL monomers.14 Only very few bacteria are able to make random copolymers of SCL and MCL monomers,15,16 so far, only one of this type of PHA, namely, a copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) has been successfully produced in large quantity for application research.15,17 PHA technology goes back to the 1970s and 1980s, when Imperial Chemical Industries (ICI) developed Biopol® PHA18 and was the first company achieving commercial production of namely PHBV. Chemie Linz AG also produced small amount of PHB for application exploitation.2 Later, Monsanto based on the ICI patents tried to produce PHA industrially but failed as the PHA produced in this ways were not competitive compared with petrochemical-based materials. Metabolix ultimately acquired ICI’s patents from Monsanto and produced P3HB4HB as early as the 1990s. They partnered with Archer Daniels Midland (ADM) for six years to produce these PHA in a capacity of 50,000 tons/year. Despite optimistic market forecasts for biopolymers including PHA with two digit growth rate expectations, ADM and Metabolix recently terminated their partnership due to the limited market success of P3HB4HB. Beginning from 1995, some Chinese companies started to trial PHA industrial production exploiting the overcapacity of the Chinese fermentation industries.2 Table 16.1 summarizes worldwide PHA producing and researching companies. Since petroleum price does not raise to a level to offset the high cost of PHA production, all the current commercially available PHA have only a limited market share of 1.4% in the bioplastic packaging market, for example.19 Figure 16.3 shows global bioplastic packaging market by product type, 2010(%).20 Therefore, more research is needed to develop technology that can significantly lower the PHA production cost to a level competitive to petroplastics. Alternatively, high value-added PHA should be developed for high-end market.

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Table 16.1  Worldwide polyhydroxyalkanoates (PHA) producing and researching companies2 Production Company Types of PHA scale (t/a) Period Applications

ICI, UK Chemie Linz, Austria btF, Austria

PHBV PHB

300 20–100

1980s to 1990s 1980s

PHB

20–100

1990s

Biomers, Germany

PHB

Unknown

1990s to present

BASF, Germany

PHB, PHBV

Pilot scale

1980s to 2005

Metabolix, USA Tepha, USA

Several PHA Several PHA

1980s to present 1990s to present

ADM, USA (with Metabolix) P&G, USA

Several PHA

Unknown PHA medical implants 50,000

2005 to present

Packaging Packaging and drug delivery Packaging and drug delivery Packaging and drug delivery Packaging and drug delivery Packaging Medical bio-implants Raw materials

1980s to 2005

Packaging

Monsanto, USA

PHB, PHBV

1990s

Raw materials

Meredian, USA Kaneka, Japan (with P&G) Mitsubishi, Japan Biocycles, Brazil Bio-on, Italy Zhejiang Tian an, China Jiangmen Biotech Ctr, China Yikeman, Shandong, China Tianjin Northern Food, China Shantou Lianyi Biotech, China Jiang su Nan Tian, China Shenzhen O’Bioer, China Tianjin Green Bio-Science (+DSM) Shandong Lukang, China

Several PHA Several PHA

Contract manufacture Plant PHA production 10,000 Unknown

2007 to present 1990s to present

Raw materials Packaging

PHB PHB PHA (unclear) PHBV

10 100 10,000 2000

1990s 1990s to present 2008 to present 1990s to present

Packaging Raw materials Raw materials Raw materials

PHBHHx

Unknown

1990s

Raw materials

PHA (unclear)

3000

2008 to present

Raw materials

PHB

Pilot scale

1990s

Raw materials

Several PHA

Pilot scale

1990s to 2005

PHB

Pilot scale

1990s to present

Packaging and medical Raw materials

Several PHA

Unknown

2004 to present

Unclear

P3HB4HB

10,000

2004 to present

Raw materials and packaging

Several PHA

Pilot scale

2005 to present

Raw materials and packaging

Several PHA

White Biotechnology for Biopolymers W,  ϭ͘ϰй ϲ͘ϳй

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Figure 16.3  Global bioplastic packaging market by product type, 2010(%). AAC, aliphatic and aromatic copolyesters; PLA, polylactic acid; PHA, polyhydroxyalkanoate; WSP, water-soluble polymers. (Pira International Ltd.)

2. STRAINS FOR PRODUCTION OF PHA Many bacteria have been found to produce PHA on different levels, for example, over 30% of soil bacteria were found to produce PHA.21 Most of these are SCL PHA, while most of the bacteria living in oil-contaminated locations, namely, oily aqueous environments or oily soils accumulate MCL PHA. Among these, few of them have been found to make copolymers of SCL and MCL monomers. Based on their growth nutrition requirements, growth rate, PHA accumulation contents etc., very few bacteria were found suitable as industrial PHA production strains (Figure 16.4). Table 16.1 summarizes all known strains used for industrial PHA productions.2 To select an ideal PHA producer, different factors have to be considered, including high substrates to PHA conversion efficiency, no pathogenesis, easy disruption of cells for PHA extraction, well-known genetics, and convenient for genetic manipulations. In addition, other factors including robust growth, not easily contaminated by phages or other bacteria, these will allow for possible continuous fermentative production of PHA (Figure 16.4). Large cell sizes are important for easy separation and more PHA accumulation although those are not always easily found. Moreover, if the strain is able to produce more than one PHA, that would be even better. Finally, PHA produced should have

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Figure 16.4 Properties required for developing suitable polyhydroxyalkanoates (PHA)-producing strains.

suitable molecular weights (Mw) for applications, and the Mw should be reproducible during different batches of fermentations (Figure 16.4). Obviously, all the production strains currently employed do not have all the good properties combined. Besides the hydrolytic cleavage of γ-butyrolactone by NaOH, 4-hydroxybutyric acid (4-HBA) and different isomers can be produced biotechnologically.22 Apart from the fact that 4-HBA is a drug (intravenous narcotic, “liquid ecstasy,” knockout drops), P4HB could also be manufactured by polycondensation starting from 4-HB.

3. PHA PRODUCED IN INDUSTRIAL SCALE PHA production involves strain development, shake flask optimization, lab and pilot fermentor studies and then industrial scale-up (Figure 16.5). Effective microbial production of PHA depends on several factors, including the final cell density, bacterial growth rate, percentage of PHA in cell dry weight (CDW), time taken to reach high final cell density, substrate to product transformation efficiency, price of substrates, and a convenient and cheap method to extract and purify the PHA (Figure 16.5).2

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Figure 16.5 Strain and process development for industrial production of polyhydroxyalkanoates (PHA).2

Figure 16.6  Polyhydroxyalkanoates (PHA) that have been produced in sufficient quantity for application research, including poly-(R)-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxyoctanoate (PHO) (up part left to right). Random copolymers of (R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate (PHBV), (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate (PHBHHx), and (R)3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) (bottom part left to right).

So far, only few PHA are produced in large scale for commercial exploitations (Figure 16.6). All of these PHA are produced using a fed-batch process to achieve high cell density growth.2 However, continuous processes are important for reducing the cost of PHA production. Increasingly, it becomes attractive to produce PHA using mixed cultures.

3.1 Fed-Batch Process For most of the SCL PHA, CDWs can reach around 100 g/L CDW after 48–60 h of fermentation containing approximately 80% PHA in CDW (Table 16.2).2 When Ralstonia eutropha is used, around 200 g/L CDW containing >80% PHA, namely PHBV can be achieved. While MCL PHA are mostly difficult to reach high cell density due to the heavy demand on

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562

Ralstonia eutropha

No

PHB

Glucose

>200

>80%

Alcaligenes latus

No

PHB

Glucose or sucrose

>60

>75%

Escherichia coli

phbCAB + vgb

PHB

Glucose

>150

>80%

R. eutropha

No

PHBV

Glucose +  Propionate

>160

>75%

R. eutropha Escherichia coli

No phbCAB

P3HB4HB

Glucose +  1,4-BD

>100

>75%

R. eutropha Aeromonas hydrophila

phaCAc No

PHBHHx PHBHHx

Fatty acids Lauric acid

>100 <50

>80% <50%

Aeromonas hydrophila

phbAB + vgb

PHBHHx

Lauric acid

∽50

>50%

Pseudomonas putida Pseudomonas oleovorans Pseudomonas entomophila Bacillus spp. Halomonas spp.

No

MCL PHA

Fatty acids

∽45

>60%

Yes

Fatty acids

>20

>70%

Sucrose Glucose

>90 >100

>50% >80%

Halomonas spp.

Yes

MCL homoPHA PHB PHB or PHBV SCL–MCL PHA

Glucose + fat

>50

>60%

No Yes

Company

Tianjin North. Food, China Chemie Linz, btf, Austria Biomers, Germany Jiangsu LanTian, China ICI, UK Zhejiang Tianan, China Metabolix, USA Tianjin Green Biosci. China P&G, Kaneka, Japan P&G, Jiangmen Biotech Ctr, China Shandong Lukang, China ETH, Switzerland Baisheng, Shandong, China Biocycles, Brazil KDN, Qingdao, China Baisheng, Shandong, China

Note: CDW: Cell dry weight; vgb: Gene encoding Vitreoscilla hemoglobin; phbCAB: PHB synthesis genes encoding β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase; Ac: Aeromonas caviae; 1,4-BD: 1,4-butanediol; phaCAc: PHA synthase gene phaC from Aeromonas caviae.

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Table 16.2  Known bacterial strains used for pilot and large-scale production of various polyhydroxyalkanoates (PHA)2 DNA Strain manipulation PHA type C-source Final CDW (g/L) Final PHA (% CDW)

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oxygen by Pseudomonas spp. that are obligate aerobes (Table 16.2). For example, when Pseudomonas entomophila was used to make PHA using fatty acids as substrates, CDW went down to around 20 g/L due to the intensive foaming problem resulted from fatty acids (Table 16.2). Reduction on aeration to avoid foaming led to reduce CDW. Although fed-batch processes are efficient to achieve high cell density fermentation in most cases, it suffers from having to interrupt the fermentation process and cleaning up the entire fermentation system including resterilization. All these operations increase the complexity of the process, leading to high PHA production cost.

3.2 Continuous Process Continuous process offers the advantages of maintaining growth conditions constantly, allowing the cells to grow to relative high density and maintaining that density for a long period of time. Since conditions are constant, CDW, PHA content, and Mw as well as PHA monomer compositions can be maintained relatively stable and reproducible during the continuous processes. However, microbial contamination is a setback especially for continuous long fermentation processes that are more prone to attract infections. To avoid contamination, it is important to select microorganisms that are robust in growth and that growth conditions selectively favor the production strains. Recently, the author’s lab found that some Halomonas spp. were able to grow to a high CDW in the presence of high salt concentrations such as 35–80 g/L NaCl and high pH of 8–11.23 Since most nonhalophilic bacteria are not able to grow under the high NaCl conditions and high pH, these PHA-producing strain grow to overdominate others. Continuous fermentation processes were conducted using the Halomonas species TD01 for at least two weeks without contaminated by other microbes. At the end of the fermentation, CDW reached over 80 g/L containing around 75% PHA. The process has been optimized by the industries in the presence of seawater instead of NaCl aqueous solution to reach over 100 g/L CDW containing over 80% of PHA (unpublished results). Most importantly, the continuous fermentation process was run for at least two weeks without bacterial contamination under 60 g/L NaCl and pH 8–9 and under open (unsterile) conditions. Such a process can save not only fresh water but also energy cost as well as intensive labors for cleaning the fermentation systems. In the future, such process should be developed to use cellulose as substrate to avoid food versus fuels or food versus chemicals disputes (Figure 16.7).

3.3 Mixed Cultures Many studies on PHA production have been focused on industrial biotechnology-based production methods using pure culture technology and genetically modified microorganisms.8 Due to the high costs for sterilizing equipment and the substrate, as well as the batch-wise processing, PHA production is complicated and expensive.

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Figure 16.7  Open, continuous, energy, and water-saving processes for production of bioplastics polyhydroxyalkanoates (PHA) and biofuels.

To lower the PHA production cost, some studies used a strategy called microbial community engineering for enrichment of PHA-producing biomass.24 They require nonsterile substrate and operational conditions. PHA-producing microorganisms selected from the natural environment, instead of a certain type of modal bacterium, are used for PHA production. Additionally, the PHA production process can be operated continuously.25 Several studies have investigated the possibility of implementing the microbial community engineering process for PHA production using synthetic wastewater. It has proven possible to enrich a stable microbial community for PHA production, while reaching a comparable productivity in terms of maximum PHA content (90% of CDW) and biomass specific production rate to pure culture process.26 The results demonstrate a possibility to produce valuable chemicals while treating wastewater. Agro-industrial waste streams instead of artificial substrates for PHA production were also used,27–29 including effluents of sugar factories, oil mills, wood mills, paper mills, or municipal wastes. However, the PHA storage capacity obtained from these studies was still significantly lower than the microbial enrichments selected on synthetic feedstock, reaching only PHA content around 55% of the dry weight.29–31 A study conducted by Albuquerque et al.25,27 obtained the currently best PHA storage capacity from real wastewater of 75% of the CDW. Nevertheless, the process should be further optimized to increase the PHA storage capacity of enrichments selected from agro-industrial wastewater.

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Figure 16.8  Polyhydroxyalkanoates (PHA) can be produced from activated sludge generated from wastewater treatments. PHA-based biofuels including 3-hydroxybutyrate methyl ester (3HBME) and 3-hydroxyalkanoate methyl esters (3HAME) can also be formed from methyl-esterifying PHA.32

PHA production from wastewater opens a new way for not only low-cost material production but also for low cost production of PHA-based biofuels including 3-hydroxybutyrate methyl ester (3HBME) and 3-hydroxyalkanoate methyl esters (3HAME) (Figure 16.8).32 Palm oil could also be a raw material resource for PHA production. Plant oils are known to generate higher PHA yields due to higher carbon content per gram of oil compared to sugars.33 Among various oils, palm oil is being studied extensively for the production of various types of PHA. It has been confirmed that high yield production of PHA could be realized from palm oil and its by-products.The studies provide preliminary results on the efficiency of palm oil bioconversion into PHA and future implementation of these substrates for PHA production systems.33

3.4 Applications of PHA PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines, and biofuels have been researched for many years. Bacterial PHA synthesis has been found

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Figure 16.9  Polyhydroxyalkanoates and its related technologies are forming an industrial value chain ranging from fermentation, materials, fine chemicals, energy to medical fields.2

to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ, PhaR, or PhaC have been found to be useful for achieving protein purification, specific drug targeting, and biosurfactants. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (Figure 16.9).2 Recently, it becomes possible to develop structurally controllable PHA for making PHA as a smart material.10,11,34

3.5 Environmentally Friendly Bioplastics for Packaging Purposes PHA were developed at the beginning as an environmentally friendly bioplastic for packaging purposes. ICI in the 1990s developed PHA with a trade name of Biopol, and was used as a shampoo bottles. Procter and Gambles (P&G) developed them with another name Nodax into a series of products ranging from coating sheets to nonwoven textiles to fibers. Metabolix trade named their PHA as Mirel. All these efforts have been targeted for bioplastic applications. In the late 1990s, many Chinese fermentation companies began to exploit their overcapacity for large-scale production of PHA (Table 16.3).2 At this time, Chinese companies are producing all types of PHA including poly-(R)-3-hydroxybutyrate (PHB),

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Table 16.3  Known companies involved in polyhydroxyalkanoates (PHA) production and application developments2 Company Types of PHA Scale (t/a) Period Applications

ICI, UK Chemie Linz, Austria

PHBV PHB

300 20–100

1980s to 1990s 1980s

BTF, Austria

PHB

20–100

1990s

Biomers, Germany

PHB

Unknown

1990s to present

BASF, Germany

PHB, PHBV

Pilot scale

1980s to 2005

Metabolix, USA Tepha, USA

Several PHA Several PHA

1980s to present 1990s to present

ADM, USA (with Metabolix) P&G, USA

Several PHA

Unknown PHA medical implants 50,000

2005 to 2012

Packaging Packaging and drug delivery Packaging and drug delivery Packaging and drug delivery Blending with Ecoflex Packaging Medical bio-implants Raw materials

1980s to 2005

Packaging

Monsonto, USA

PHB, PHBV

1990s

Raw materials

Meredian, USA Kaneka, Japan (with P&G) Mitsubishi, Japan Biocycles, Brazil Bio-on, Italy Zhejiang Tian an, China Jiangmen Biotech Ctr, China Yikeman, Shandong, China Tianjin Northern food, China Shantou Lianyi, China

Several PHA Several PHA

Contract manufacture Plant PHA production 10,000 Unknown

2007 to present 1990s to present

Raw materials Packaging

PHB PHB PHA (unclear) PHBV

10 100 10,000 2000

1990s 1990s to present 2008 to present 1990s to present

Packaging Raw materials Raw materials Raw materials

PHBHHx

Unknown

1990s

Raw materials

PHA (unclear) PHB

3000

2008 to present

Raw materials

Pilot scale

1990s

Raw materials

Several PHA

Pilot scale

1990s to 2005

Jiangsu Nan Tian, China Shenzhen O’Bioer, China Tianjin Green Bio-Science, China Shandong Lukang, China Qingdao VLand, China Shandong Baisheng, China

PHB

Pilot scale

1990s to present

Packaging and medicals Raw materials

Several PHA

Unknown

2004 to present

Unclear

P3HB4HB

10,000

2004 to present

Raw materials

Several PHA

Pilot scale

2005 to present

Raw materials

Several PHA Several PHA

Pilot scale Pilot scale

2012 to present 2012 to present

Raw materials Raw materials

Several PHA

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poly-4-hydroxybutyrate (P4HB), poly-3-hydroxyoctanoate (PHO) (up part left to right), random copolymers of (R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate (PHBV), (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate (PHBHHx), and (R)3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) (Figure 16.6). However, due to the high cost of production, PHA as an environmentally friendly plastic still have a very limited market. Much more efforts are still needed to lower the production cost to a level comparable to the petrochemical-based plastics.

3.6 Biofuels HAME derived from bacterial PHA was developed by the author’s lab,32 the PHA-based biofuel could provide a strong candidate for current biofuels or fuel additives market. The term biofuels is used for all types of biomass-derived fuels employed in the transportation sector. Most biofuels can be utilized either as a substitute for common fossil fuels such as gasoline and diesel or in blends with them. Other such as biomass-based natural gas requires changes in both vehicle construction and fuel distribution infrastructure.35 PHA universally synthesized by many bacteria grown in various carbon sources including wastewater that can be cleaved and reacted with methanol to form their corresponding methyl esters.32 3HBME or 3HAME improved the combustion heat of ethanol with 3HAME having more obvious effects. With the increased content of 3HBME or 3HAME in the ethanol, the combustion heats of blended fuels remained at the similar levels. This may enhance the possibility to use 3HAME as a biofuel because the addition of a low content on 3HAME can have similar effects.32 Since PHA can be obtained from wastewater treatment process, the PHA cost can be significantly reduced for biofuels purpose. Fuel ethanol has a combustion heat (CH) of over 26 kJ/g, higher than 21 kJ/g of 3HBME (Table 16.4), both of their CH are approximately half of that of gasoline.When blended with HBME, all CH showed a slight reduction, ranging from 2 to 6 kJ/g, depending on the amount of HBME or ethanol added to the gasoline (Table 16.4). For ethanol–gasoline blends, their CH decreased with increasing ethanol ratios in the blends, all the CH of the blends were lower than that of the pure gasoline, the highest CH of Table 16.4  Heats of combustion from blends of HBME–gasoline compared with that of ethanol–gasoline30 Sample Combustion heat (MJ/kg) Sample Combustion heat (MJ/kg)

HBME H5 H8.5 H10 H15 H20

21.11 ± 0.41 36.90 ± 0.52 37.34 ± 0.02 37.28 ± 0.09 36.54 ± 0.12 34.52 ± 0.25

Ethanol E5 E10 E15 E20 97#Gasoline

26.21 ± 0.10 38.66 ± 0.02 38.51 ± 0.23 38.43 ± 0.24 36.79 ± 0.23 40.07 ± 0.78

H denotes HBME–gasoline blends, E is ethanol–gasoline blends, and the next number stands for the volume ratio. For example, E5 means 5% (VV-1) HBME in gasoline.

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the blend (E5) can be 96.5% of gasoline. However, for HBME–gasoline blends, the CH of the blends showed firstly a decrease, then increase to reach the maximum at nearly 8.5% before the CH decreased again at H15.36 These results clearly show that HBME, when mixed with gasoline in ratios of 8.5–10 (v/v)%, could have a positive synergistic effort on gasoline combustion as revealed by fuel related properties shown below, and this explains why reduction on CH for HBME– gasoline blends was less serious compared with that of ethanol–gasoline blends. PHA obtained from wastewater can also be cleaved and reacted with methanol to form their corresponding methyl esters. The resulting 3HBME and 3HAME improved the combustion heat of ethanol, with 3HAME having more obvious effects. In addition, 3HBME and 3HAME could also be used as fuel additives for other fuels such as propanol, butanol, gasoline, and diesel. Because the application of PHA does not require highly purified PHA, the production process appears to be much simpler.32 Therefore, the application of PHA as a biofuel allows the PHA industry to become a close cycle (Figure 16.10).

3.7 Medical Implants PHA have been studied for medical implants applications such as heart valves, vascular tissues, bone tissues, cartilage replacements, nerve conduits, as well as esophagus tissues. Many studies have been carried out in the author’s lab in the past 20+ years. Recently, mechanism on PHA-promoted tissue regeneration was revealed to relate cell responses to PHA biodegradation products and cell–material interactions mediated by microRNA.34,37 Very importantly, PHA implants were found not to cause carcinogenesis during long-term implantation.38 PHA have demonstrated great potentials in biomedical areas.39

Figure 16.10  Polyhydroxyalkanoates (PHA) production and application as bioplastics and biofuels will close the PHA application cycle, leading to reduced CO2 emission. Left picture: a1 and b1, combustion of fuel ethanol; b1 and b2, combustion of HBME.36

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3.8 Monomers as Chiral Intermediates As monomers of PHA, chiral 3-hydroxyalkanoic acids (3HAs) are important chemicals used as precursors or intermediates for the synthesis of various fine compounds including pharmaceuticals, antibiotics, food additive, fragrances, and vitamins.3,40,41 With over 150 different monomers, PHA are a rich source of chiral hydroxyalkanoic acids. PHA monomers can be divided into two groups according to their carbon-chain length: SCL 3HA contains 3–5 carbon atoms and MCL 3HA contains 6–14 carbon atoms. MCL 3HA have shown potential to be pharmaceuticals of high value.42 For example, 3-hydroxyhexanoic acid (3HHx) can be used as intermediate for synthesizing analogs of laulimalide, an anticancer chemical.43 3-hydroxyoctanoic acid (3HO) exhibits potential antimicrobial activities44; 3HO is also an intermediate for statins known as HMG-CoA reductase inhibitors.45 Myrmicacin, identified as 3-hydroxydecanoic acid (3HD), was found as a growth inhibitor against pollen, fungi, and bacteria.46,47 3-hydroxydodecanoic acid (3HDD) and 3-hydroxytetradecanoic acid (3HTD) are common constituent of lipid A in the cell surface of Gram-negative bacteria.48

3.9 Smart Materials The application of PHA as a low-cost biodegradable plastic has been hampered by its higher production cost and the difficulty to precisely control their structures and properties. Global efforts have been made to develop technology for lowering the PHA production cost. With the successful construction of β-oxidation weakened Pseudomonas spp. as PHA production platforms, we are now able to control the formation of homopolymers, random and block copolymers including monomer structures and ratios, this allows us to obtain PHA with consistent properties. At the same time, we can introduce various functional groups into the PHA site chains in a quantitative way, which provide more opportunities for site chain grafting. Functional PHA together with endless possibilities for grafting have provided us limitless ways of making new PHA, possibly with some high value-added functionalities, leading to smart materials.

3.10 Challenges for R&D To develop PHA into a commercially acceptable material, following efforts have to be made: first, feasible technology must be developed to reduce PHA production cost to a level competitive to petroleum plastics; secondly, value-added functionalities must be provided to PHA so that high production cost can be compensated by its high-end-applications. To reduce PHA production cost, following efforts should be made: 1. Achieving high cell density fermentation with high PHA content in CDW: This is important as a high cell density will allow effective cells and PHA recovery; 2. Grow cell in low cost substrates (or mixed substrates): Substrates contribute 50–60% of PHA production cost, low cost substrates such as sludge hydrolyzed products or

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kitchen waste, or even hydrolyzed cellulose (if cheap), can be consumed by many PHA-producing organisms; 3. Grow cell in unsterile process in mixed substrates: Sterilization is an energy consuming process, maintaining sterilization throughout the fermentation process is costly and complicated; 4. Grow cell in continuous process in mixed substrates under unsterile conditions:This is a close to ideal situation, in which, if succeeded, will significantly save energy, reduce process complexity, and increase production efficiency; 5. Achieve ultrahigh PHA accumulation in the cells in continuous process in mixed substrates: If the cells contain over 95% PHA in their CDWs, the washing process will increase PHA content further to over 98% content, this ultrahigh PHA content provides a possibility to process the dry or even wet cells directly as plastic pellets in extruders; 6. Increase substrates to PHA conversion efficiency: Many substrates are consumed by the cells to maintain their metabolic activities, these substrates are often turned into CO2 and water. If the waste of substrates can be reduced, it helps to reduce production cost; 7. Achieve controllable cell flocculation for easy separation after continuous growth in mixed substrates: Downstream processing contributes 30–50% of PHA cost, controllable flocculation reduces cells and broth separation cost, thus leading to reduction on PHA production cost; 8. Achieve controllable PHA synthesis (homopolymers, random and block copolymers, functional polymers) in large scale: Microbial processes often produce inconsistent ratios of monomers in PHA copolymers. This significantly affects PHA applications. Technology must be developed to control the PHA micro and macrostructures so that all microbial PHA have consistent structures and properties; 9. Production and applications of chiral PHA monomers: Chiral PHA monomers are mostly difficult to make chemically. If a large-scale application on certain PHA monomers can be found, it will increase the PHA values; 10. Turning low cost PHA into functional PHA:This will save a lot of efforts to reduce PHA production cost. To achieve functional PHA, applications must be identified that make good use of unique PHA properties including biodegradability, biocompatibility, high Mw, and ease of making block copolymerization.

3.11 Future Prospective At this moment, PHA are far less competitive compared with its petrochemical peers. Beside its relative poor properties, high cost is the major factor preventing its successful large-scale marketing. Effects have been made by many labs around the globe to address the 10 challenges listed above. We foresee that ultrahigh PHA production strains with controllable flocculation properties are successfully grown to high cell density (>200 g/L) in a continuous

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fermentation process using low cost substrates including hydrolyzed cellulose, hydrolyzed sludge, or kitchen waste under unsterile conditions. PHA produced by such strains under these robust conditions have consistent structures and thus stable properties. The PHA produced in the above way have a cost similar to petroleum plastics, allowing PHA to compete with conventional plastics not only with its environmentally friendly features but also with its low cost and sustainability. By then PHA will have a significant share of the big pie belonging to conventional packaging materials. On the other hand, smart PHA materials with properties of shape memory, selective gas permeability, selective liquid permeability, temperature and/or pH responsibility, self-healing, controllable degradation and/or controllable porosity will be developed. These properties will add values to the microbial PHA, they should have a small but big margin market.

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