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ScienceDirect Polyhydroxyalkanoates, challenges and opportunities Ying Wang1, Jin Yin1 and Guo-Qiang Chen Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based plastics. Addresses MOE Key Lab of Bioinformatics, School of Life Science, TsinghuaPeking Center for Life Sciences, Tsinghua University, Beijing 100084, China Corresponding author: Chen, Guo-Qiang ([email protected]) 1 Equal contributors.
Current Opinion in Biotechnology 2014, 30:59–65 This review comes from a themed issue on Chemical biotechnology
monomer variations [5,6]. Therefore, PHA have a wider material properties. Common PHA monomers are 3-hydroxybutyrate (3HB or C4), 3-hydroxyvalerate (3HV or C5), 3-hydroxyhexanoate (3HHx or C6), 3-hydroxyoctanoate (3HO or C8), 3-hydroxydecanoate (3HD or C10) and 3-hydroxydodecanoate (3HDD or C12) as well as 4-hydroxybutyrate (4HB) [6,7]. They can be used to form homopolymers, random copolymers or block copolymers [8,9,10,11], leading to diversity of material properties. Many companies have been set up to commercialize PHA as biodegradable plastics [1]. PHA have been marketed as environmentally friendly bioplastics with less CO2 emission and sustainability as well as independence from petroleum sources [4]. However, large-scale marketing on PHA has been less successful due to the high production cost [1], inconsistent properties as well as difficulties in plastic processing compared with mature conventional plastics from petroleum source [12,13]. More efforts must be made to make PHA as competitive as conventional plastics, especially in production cost. This paper reviews challenges and recent progresses on developing technologies to lower PHA production cost or increase their values, pointing to the future technology developments to make PHA as competitive as conventional plastics.
Edited by Curt R Fischer and Steffen Schaffer
Challenges Economic situation and material properties http://dx.doi.org/10.1016/j.copbio.2014.06.001 0958-1669 # 2014 Published by Elsevier Ltd. All right reserved.
Introduction Polyhydroxyalkanoates (PHA), a family of biodegradable and biocompatible polyesters, have been produced in different scales via fermentation processes as environmentally friendly and sustainable plastics [1,2,3]. Unlike other biobased polymers such as polylactide or poly(lactic acid) termed PLA, poly(butylene succinate) short as PBS, poly(propylene carbonate) or PPC, polytrimethylene terephthalate (PTT), bio-polyethylene (bio-PE), bio-polypropylene (bio-PP), bio-poly(ethylene terephthalate) (bio-PET) [4], PHA are completely biosynthesized and biopolymerized, possessing over 150 www.sciencedirect.com
Following factors summarize some big challenges that PHA industry faces: first, PHA production is still a complicated process with a low efficiency, resulting in high production cost [14]; second, petroleum, as a raw material for conventional plastics, will not increase its price dramatically due to the recent successful exploitation of shale gas [15]; third, glucose coming from hydrolysis of starch, also a major raw material for PHA production, has increased its price very fast [16]; fourth, PHA do not have very consistent structures and properties compared with their competitors, petrochemical plastics [17]; fifth, PHA processing is more difficult than conventional plastics due to their slow crystallization process [12,18]; sixth, we have yet to develop high value added applications for PHA [19]. Technological challenges
Chemical industry has been producing low cost plastics for the material industries for many decades. In comparison, biotechnology is still an expensive industry, leading Current Opinion in Biotechnology 2014, 30:59–65
60 Chemical biotechnology
Table 1 Comparisons between bioprocessing and chemical processing Comparison parameter
Biotechnology
Chemical technology
Raw materials Reaction conditions
Sustainable agriculture resources including CO2 Ambient temperature and atmosphere pressure, aqueous medium
Process Process duration
Mostly discontinuous batch processes From inoculation to fermentations to downstream take one to two weeks High due to sterilization and continuous aeration Heavy water consumption Mostly low, from mg up to 200 g/L Very high Low
Petroleum Mostly high temperature, high pressure and organic solvents as reaction medium Mostly continuous processes Mostly completed within days
Energy consumption Water consumption Final product concentration Cost of product recovery Substrate to product conversion efficiency Risk
Waste water
Low level
Mostly nontoxic and easily treated
to more expensive bio-based chemicals, materials (bioplastics) and biofuels [20]. It is therefore important to compare the advantages and disadvantages of bioprocessing and chemical processing, so that relevant technology can be developed to make bioprocessing more competitive (Table 1). Biotechnology or bioprocessing has some clear advantages over the chemical counterpart (Table 1), including the use of sustainable resources such as starch, cellulose, fatty acids even CO2. At the same time, bioprocess is normally carried out at ambient temperature and atmosphere pressure in aqueous media. The resulting wastewater is generally nontoxic and easily treated. Unlike chemical processing, risks associated with fires and explosions are minimal [21]. However, the disadvantages of bioprocessing are also obvious: most fermentation (bioprocessing) processes are discontinuous ones, they take one to several weeks to complete from inoculum to downstream product purification. Bioprocessing consumes a lot of precious fresh water, the sterilization and aeration processes also demand a lot of energy, adding to the cost of final products. In addition, final products in fermentation broths are generally very low, ranging from mg to 200 g/L. The lower yield leads to a high downstream purification cost. By contrast, chemical industry can reach at least 500 g/L at the end of the process. Most seriously, the substrate to product conversion efficiency is very low in fermentation. In PHA industry, the most common substrate to PHA conversion stands around 1/3 or 33% (g/g) [22], while the chemical synthesis of plastics such as PE, PET or PS, etc. can be as high as over 90% or even close to 100% (Table 1) [23]. Therefore, to make bio-products competitive, we must learn from the advantages of chemical industries while Current Opinion in Biotechnology 2014, 30:59–65
Depending on products Less water consumption Mostly over 500 g/L Low Mostly very high High due to flammable and explosive, as well as toxic gas or product leakages Mostly toxic, acidic or alkali, difficult to treat
maintaining the advantages of bioprocessing. Technology should be developed to address the setbacks on bioprocessing. Opportunities
Chemical processing has been a very effective industry for the past decades. Efforts have been made in biotechnology research communities to get close to the advantages of chemical industry. Recent advancements in systems and synthetic biology allow construction of a super PHA production strain to satisfy our many technology demands for lowering cost PHA production. Construction of a super PHA production strain
An industrial production strain, especially for white biotechnology, should possess the following properties: not a pathogen, no phage, clear genomic background, easy genomic manipulation, no toxin production, fast growth in a mineral medium (MM) and cellulose utilization (if possible) (Figure 1). Ideally, the strain should also have a wider temperature and pH window for fast growth. With these properties available, it is not so difficult to construct other desirable properties for a low cost PHA production such as high PHA accumulation level (over 90% cell dry weight), starch or even mixed substrate utilization, a high substrate to PHA conversion efficiency (over 50% g/g), and ability for production of short-chain-length (scl) and/or medium-chain-length PHA. For convenient downstream PHA recovery and purification, the above super strain should also have a large cell size, fragile cell wall, easy or inducible flocculation, etc. With many knowledge available today, it is a matter of time that the ideal and super PHA production is available for low cost PHA production (Figure 1 and Table 2). Further genetic manipulation allows the development of additional advantages for mimicking the chemical processing (Table 2). www.sciencedirect.com
Polyhydroxyalkanoates, challenges and opportunities Wang, Yin and Chen 61
Figure 1
Easy flocculation Fas t In M growth Mm edia
Hi acc gh PH um A ula tion
Growth at high or low pH Large size
No phage
Growth scl-and mcl producer at high or low temp
Cellulose utilizer
Fragile cell wall
No toxin
Clear genomic background Growth in mixed carbon sources High substrate to PHA conversion
No pathogen Easy genomic manipulation Starch utilizer
Current Opinion in Biotechnology
Desirable properties for a PHA industrial production strain.
To achieve a high final product concentration similar to chemical processing (Table 1), cell density should be reached as high as possible (>200 g/L), PHA content should be as high as over 90% or even to 95% of the cell dryweight (CDW) (Table 2). These goals can be realized by manipulating genes related to quorum sensing, oxygen
uptake and PHA synthesis mechanisms [24]. Since oxygen is always a limiting factor for high cell density growth, a separation of cell growth followed by an oxygen limitation induced PHA accumulation stage will be an ideal situation to maximize the PHA growth accompanied by maximal PHA yield. This separation on growth and
Table 2 Technology to be developed to lower PHA production cost Technology
Reasons and/or purpose
High cell density fermentation
Achieve effective growth and cells recovery
Growth cells in low cost substrates or mixed substrates Fast growing cells
Substrates contributed to over 60% of PHA cost
Fast growing CO2 utilizing bacteria able to produce PHA Open (unsterile) and continuous fermentation process PHA synthesis induced by oxygen limitation Ultrahigh PHA accumulation (over 95% PHA in cell dry weight) Increase substrate (mostly carbon sources) to PHA conversion efficiency Enlarging the PHA production cells Inducible cell flocculation Inducible cell lysis Cell disruption by PHA hyperproduction Extracellular PHA production
Reduce fermentation duration and avoid microbial contamination CO2 is a free substrate To save sterilization energy, reduce fermentation complexity and improve process effectiveness Oxygen is a limited factor in all high cell density growth To avoid expensive and complicated downstream PHA purification process Substrates contributed to over 60% of PHA cost
To allow more cellular space for PHA accumulation, this also allows easy cells recovery Allow easy biomass recovery after fermentation Allow easy PHA granules recovery after biomass harvest Save the biomass harvest process
Large PHA granules
Not limited by a small cellular space, also for easy PHA granule recovery Allow easy recovery of PHA granules from lysis broth
A synthetic cell combining the above properties
Achieve up-stream and down-stream competitivenesses
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Methodology Manipulation on quorum sensing and cell oxygen uptake mechanisms Screening targeted substrates utilizing bacteria able to produce high content PHA Minimizing bacterial genome, changing cell growth patterns Manipulating the CO2 uptake mechanism such as carboxysomes, etc. Screening for PHA producers able to grow fast in extreme environments such as high or low pH and temperature, high osmotic pressure, etc. Place PHA synthesis operons behind microaerobic promotor Manipulating the PHA synthesis mechanism and PHA synthases Removing pathways that consume substrates for non-PHA metabolisms, and/or reinforce PHA synthesis flux Engineering the cell division patterns and/or cytoskeletons Inducible expression of surface displaying adhesive proteins Inducible expression of cell lysis proteins Manipulating the PHA synthesis mechanism and PHA synthases Need new PHA synthesis mechanisms Manipulating the formation of PHA granules associated proteins An artificial cell with assembled functional DNA
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PHA production can be materialized by placing PHA synthesis operons behind microaerobic promotor [25]. Since substrates play the most important part in PHA cost, it is crucial to use low cost substrates such as mixed substrates (kitchen wastes), low cost starch, cellulose (and/or its hydrolyzed products) or even CO2. It is normally easier to isolate mixed substrates utilizing microorganisms than engineering them. However, it has become possible to manipulate the CO2 uptake mechanism such as carboxysomes, etc. to take up CO2 as substrate for cell growth [26,27]. Also, a combination of deletions on pathways that consume PHA monomers and over-expressions of PHA production pathways can lead to increase substrates to PHA conversion efficiency (Table 2) [28]. Development of competitive PHA production processes
Downstream processing of biological products is also costly. For example, it requires continuous centrifugation process to separate PHA containing cells from the fermentation broth [1]. While the cells are small and light with lower concentration in the broth, separation via continuous centrifugation is a long and energy intensive process. It is therefore important to enlarge the cell sizes, and/or construct inducible cell flocculation to ease the precipitation process. Systems and synthetic biology allow creation of pathways in PHA production bacteria combining cell division and surface display of adhesive proteins (Table 2). After cells separation from the broth, inducible cell lysis is important to release the intracellular PHA granules [29], this can be achieved by induced expression of lysis proteins such as lysozymes, exonucleases and proteinases, etc. Ideally, PHA synthesis mechanism should be exploited so that ultrahigh PHA production up to 95% in CDW can be reached. Such a high PHA accumulation level can lead to cell ruptures, easing the cell lysis process. PHA granules should be large in size for easy separation. By manipulating the PHA granule associated proteins, it is possible to increase PHA granule size [30]. Extracellular production of PHA will be an ideal way of making PHA since extracellular PHA accumulation is not limited by the small cell volume and is convenient for downstream extraction [31]. This points to a future with unlimited production of PHA via extracellular mechanism. For example, bacterial cell walls can be weakened by deleting some cell wall synthesis related genes. According to some related studies recently conducted in this lab, PHA could be produced extracellularly via weakened cell walls for the benefit of enhanced PHA production and extraction. The above cell properties including large cell size, ultrahigh PHA accumulation, inducible flocculation and lysis, large PHA granules, extracellular PHA synthesis and high cell density growth can be achieved by genetic Current Opinion in Biotechnology 2014, 30:59–65
engineering, pathway engineering and synthetic biology. There is still one setback of the current PHA production process, which is its discontinuity requiring energy intensive sterilization using high-pressure hot steam. To overcome this difficulty, PHA should mimic chemical processing using continuous and unsterile processes. Recently, it has become possible to recruit mixed cultures for continuous and unsterile production of PHA [32]. However, pure culture is preferred as it usually lead to more reproducible results. Fortunately, halophilic bacteria offer such possibilities as they grow in medium to high salt concentrations under high pH in a wide temperature window, such growth conditions can effectively prevent contamination by other microorganisms [33]. In the authors’ lab, we are able to grow halophilic bacteria in unsterile and continuous process in seawater like medium for at least one month without any microbial contamination [33], the halophilic bacteria have been proven easy for genetic manipulation, allowing the construction of above mentioned super PHA production strains [34]. Since the halophilic strains can grow in seawater in a continuous and unsterile way, the development of these types of strains for PHA industrial production can save a lot of fresh water and energy, reduce process complexity and thus, increase the competitiveness of PHA. High value added PHA production and applications
PHA that have been produced in large scale including poly(3-hydroxybutyrate) (PHB) [15], poly(4-hydroxybutyrate) (P4HB) [35,36], copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) [1], copolymers of 3hydroxybutyrate and 3-hydroxyvalerate (PHBV) [1], as well as similar copolymers of 3-hydroxybutyrate and 3hydroxyhexanoate (PHBHHx) [1]. Except the application as biomedical implant materials, all the above PHA are mainly developed as environmentally friendly biodegradable packaging materials which are low value added. To exploit the low value added yet expensive PHA materials produced in large scale, it is important that the applications can generate high value and quantity demand is large. Recently, high quality textile from PHBV has been successfully manufactured (Table 3). If the marketing is successful, this will open a large quantity market for PHA. On the other hand, it was reported that ultrahigh molecular weight PHA can be used to make ultrastrong fibers for fishing lines and fishing net, etc. [37,38]. The ultra strong property is also a good sale for high value. Recently, the authors’ lab succeeded in engineering the b-oxidation pathway encoded on the chromosomes of Pseudomonas putida and Pseudomonas entomophiles, allowing the synthesis of controllable PHA microstructures including formation of PHA homopolymers, composition www.sciencedirect.com
Polyhydroxyalkanoates, challenges and opportunities Wang, Yin and Chen 63
Table 3 Technology to be developed to increase PHA values Technology Controllable PHA synthesis with defined structures
Controllable formations of functional PHA
Increase PHA diversity Ultrahigh molecular weight Production of single chiral PHA monomers
PHA as medical implant materials PHA as good quality textiles
Reasons and/or purpose To form PHA homopolymers, defined ratios monomers in random copolymers and block copolymers To form PHA smart materials with properties of pH, temp, and wettability sensitivities or shape memory, etc. To find other possible applications To obtain ultrastrong PHA fibers Mostly as drugs or as lead compounds for drug molecule synthesis PHA are biodegradable and biocompatible polyesters PHA can be turned into textiles that have values higher than packaging materials
adjustable random copolymers and block copolymers [10,39,40]. Since PHA structures can be designed, we can obtain required material properties based on precisely designed and synthesized PHA. Especially on the block copolymers reported to have anti-aging property [41], a series of diblock copolymers including P3HB-b-P4HB [42], P3HB-b-P3HHx [43], and P3HP-b-P4HB [8] were successfully synthesized and found to have one or two improved properties over their two relative homopolymers, random copolymers or blend polymers. Most importantly, block ratios in copolymers can be adjusted, leading to adjustable polymer properties. Since functional groups can be inserted into PHA chains, such as double or triple bonds, epoxy, carbonyl, cyano, phenyl and halogen [44], graft PHA polymers can be synthesized by chemical modification of the PHA side chains, leading to dramatic changes in PHA properties. There are limitless possibilities to create new graft PHA homopolymers or copolymers. Finally, PHA monomers are mostly chiral hydroxyalkanoic acids (HA), microbial production of chiral HA can be achieved by deletions of partial b-oxidation pathway and PHA synthase phaC operon combined with over-expression of thioesterase genes in Pseudomonas entomophila [45], the resulting recombinant P. entomophila was able to produce various chiral HA, respectively, from their related carbon sources. Since chiral HA are important chemicals used as precursors or intermediates for the synthesis of various fine compounds including pharmaceuticals, antibiotics, food additive, fragrances, and vitamins [3,5], they have high value added applications waiting to be exploited. Future prospects
The recent advances on systems and synthetic biology will allow the design and construction of a PHA super www.sciencedirect.com
Methodology Engineering the PHA synthesis pathways, especially the b-oxidation pathways Additions of functional PHA as monomer precursors
Chemical grafting of PHA functional side chains Engineering the PHA synthase or operon Deleting PHA synthases in PHA synthesis pathway PHA can be turned into any shape for implant purposes Controllable crystallization of PHA fibers
production strain that will be able to grow fast to high cell density (>200 g/L) utilizing low cost substrates including cellulose, starch or even kitchen wastes under a very high carbon source to PHA conversion efficiency of at least 50% (g/g). The synthetic cell will be able to achieve oxygen limitation induced >90% PHA accumulation in cell dry weights. After completing the PHA production, the very large cells will be induced for flocculation precipitation followed by induced cell lysis to release large PHA granules. The PHA fermentation process will be conducted under unsterile and continuous way using seawater to save energy and fresh water, reduce process complexity so that the resulting PHA can be competitive in production cost with petroleum based plastics. The above super PHA production strain will be constructed to allow precisely control their PHA structures to form homopolymers, random copolymers and block copolymers as well as functional polymers with precise monomer structures and ratios for consistent properties. High value added applications based on unique PHA polymer properties and chiral hydroxyalkanoic acids will be developed for the high end markets.
Acknowledgements This project has been supported by State 973 Basic Science Research Project (Grant No.: 2012CB725201) and Natural Science Foundation of China (Grant No. 31270146).
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An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. This process opens a new area for reducing the cost in polyhydroxyalkanoates production.
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41. Pederson EN, McChalicher CW, Srienc F: Bacterial synthesis of PHA block copolymers. Biomacromolecules 2006, 7:1904-1911. 42. Hu D, Chung AL, Wu LP, Zhang X, Wu Q, Chen JC, Chen GQ: Biosynthesis and characterization of polyhydroxyalkanoate block copolymer P3HB-b-P4HB. Biomacromolecules 2011, 12:3166-3173. 43. Tripathi L, Wu LP, Chen J, Chen GQ: Synthesis of diblock copolymer poly-3-hydroxybutyrate-block-poly-3hydroxyhexanoate [PHB-b-PHHx] by a beta-oxidation weakened Pseudomonas putida KT2442. Microb Cell Fact 2012, 11:44. 44. Olivera E, Arcos M, Naharro G, Luengo J: Unusual PHA biosynthesis. In Plastics from Bacteria. Edited by Chen GQ. Berlin/Heidelberg: Springer; 2010:133-186. 45. Chung AL, Zeng GD, Jin HL, Wu Q, Chen JC, Chen GQ: Production of medium-chain-length 3-hydroxyalkanoic acids by b-oxidation and phaC operon deleted Pseudomonas entomophila harboring thioesterase gene. Metab Eng 2013, 17:23-29. A metabolic engineered pathway was constructed in Pseudomonas entomophila L48 to produce MCL 3HA. For the first time the paper revealed that fine chemicals 3HTD, 3HDD or 3HD could be efficiently produced by metabolic engineered b-oxidation in Pseudomonas spp. grown on related fatty acids.
Current Opinion in Biotechnology 2014, 30:59–65
ScienceDirect Polyhydroxyalkanoates, challenges and opportunities Ying Wang1, Jin Yin1 and Guo-Qiang Chen Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based plastics. Addresses MOE Key Lab of Bioinformatics, School of Life Science, TsinghuaPeking Center for Life Sciences, Tsinghua University, Beijing 100084, China Corresponding author: Chen, Guo-Qiang ([email protected]) 1 Equal contributors.
Current Opinion in Biotechnology 2014, 30:59–65 This review comes from a themed issue on Chemical biotechnology
monomer variations [5,6]. Therefore, PHA have a wider material properties. Common PHA monomers are 3-hydroxybutyrate (3HB or C4), 3-hydroxyvalerate (3HV or C5), 3-hydroxyhexanoate (3HHx or C6), 3-hydroxyoctanoate (3HO or C8), 3-hydroxydecanoate (3HD or C10) and 3-hydroxydodecanoate (3HDD or C12) as well as 4-hydroxybutyrate (4HB) [6,7]. They can be used to form homopolymers, random copolymers or block copolymers [8,9,10,11], leading to diversity of material properties. Many companies have been set up to commercialize PHA as biodegradable plastics [1]. PHA have been marketed as environmentally friendly bioplastics with less CO2 emission and sustainability as well as independence from petroleum sources [4]. However, large-scale marketing on PHA has been less successful due to the high production cost [1], inconsistent properties as well as difficulties in plastic processing compared with mature conventional plastics from petroleum source [12,13]. More efforts must be made to make PHA as competitive as conventional plastics, especially in production cost. This paper reviews challenges and recent progresses on developing technologies to lower PHA production cost or increase their values, pointing to the future technology developments to make PHA as competitive as conventional plastics.
Edited by Curt R Fischer and Steffen Schaffer
Challenges Economic situation and material properties http://dx.doi.org/10.1016/j.copbio.2014.06.001 0958-1669 # 2014 Published by Elsevier Ltd. All right reserved.
Introduction Polyhydroxyalkanoates (PHA), a family of biodegradable and biocompatible polyesters, have been produced in different scales via fermentation processes as environmentally friendly and sustainable plastics [1,2,3]. Unlike other biobased polymers such as polylactide or poly(lactic acid) termed PLA, poly(butylene succinate) short as PBS, poly(propylene carbonate) or PPC, polytrimethylene terephthalate (PTT), bio-polyethylene (bio-PE), bio-polypropylene (bio-PP), bio-poly(ethylene terephthalate) (bio-PET) [4], PHA are completely biosynthesized and biopolymerized, possessing over 150 www.sciencedirect.com
Following factors summarize some big challenges that PHA industry faces: first, PHA production is still a complicated process with a low efficiency, resulting in high production cost [14]; second, petroleum, as a raw material for conventional plastics, will not increase its price dramatically due to the recent successful exploitation of shale gas [15]; third, glucose coming from hydrolysis of starch, also a major raw material for PHA production, has increased its price very fast [16]; fourth, PHA do not have very consistent structures and properties compared with their competitors, petrochemical plastics [17]; fifth, PHA processing is more difficult than conventional plastics due to their slow crystallization process [12,18]; sixth, we have yet to develop high value added applications for PHA [19]. Technological challenges
Chemical industry has been producing low cost plastics for the material industries for many decades. In comparison, biotechnology is still an expensive industry, leading Current Opinion in Biotechnology 2014, 30:59–65
60 Chemical biotechnology
Table 1 Comparisons between bioprocessing and chemical processing Comparison parameter
Biotechnology
Chemical technology
Raw materials Reaction conditions
Sustainable agriculture resources including CO2 Ambient temperature and atmosphere pressure, aqueous medium
Process Process duration
Mostly discontinuous batch processes From inoculation to fermentations to downstream take one to two weeks High due to sterilization and continuous aeration Heavy water consumption Mostly low, from mg up to 200 g/L Very high Low
Petroleum Mostly high temperature, high pressure and organic solvents as reaction medium Mostly continuous processes Mostly completed within days
Energy consumption Water consumption Final product concentration Cost of product recovery Substrate to product conversion efficiency Risk
Waste water
Low level
Mostly nontoxic and easily treated
to more expensive bio-based chemicals, materials (bioplastics) and biofuels [20]. It is therefore important to compare the advantages and disadvantages of bioprocessing and chemical processing, so that relevant technology can be developed to make bioprocessing more competitive (Table 1). Biotechnology or bioprocessing has some clear advantages over the chemical counterpart (Table 1), including the use of sustainable resources such as starch, cellulose, fatty acids even CO2. At the same time, bioprocess is normally carried out at ambient temperature and atmosphere pressure in aqueous media. The resulting wastewater is generally nontoxic and easily treated. Unlike chemical processing, risks associated with fires and explosions are minimal [21]. However, the disadvantages of bioprocessing are also obvious: most fermentation (bioprocessing) processes are discontinuous ones, they take one to several weeks to complete from inoculum to downstream product purification. Bioprocessing consumes a lot of precious fresh water, the sterilization and aeration processes also demand a lot of energy, adding to the cost of final products. In addition, final products in fermentation broths are generally very low, ranging from mg to 200 g/L. The lower yield leads to a high downstream purification cost. By contrast, chemical industry can reach at least 500 g/L at the end of the process. Most seriously, the substrate to product conversion efficiency is very low in fermentation. In PHA industry, the most common substrate to PHA conversion stands around 1/3 or 33% (g/g) [22], while the chemical synthesis of plastics such as PE, PET or PS, etc. can be as high as over 90% or even close to 100% (Table 1) [23]. Therefore, to make bio-products competitive, we must learn from the advantages of chemical industries while Current Opinion in Biotechnology 2014, 30:59–65
Depending on products Less water consumption Mostly over 500 g/L Low Mostly very high High due to flammable and explosive, as well as toxic gas or product leakages Mostly toxic, acidic or alkali, difficult to treat
maintaining the advantages of bioprocessing. Technology should be developed to address the setbacks on bioprocessing. Opportunities
Chemical processing has been a very effective industry for the past decades. Efforts have been made in biotechnology research communities to get close to the advantages of chemical industry. Recent advancements in systems and synthetic biology allow construction of a super PHA production strain to satisfy our many technology demands for lowering cost PHA production. Construction of a super PHA production strain
An industrial production strain, especially for white biotechnology, should possess the following properties: not a pathogen, no phage, clear genomic background, easy genomic manipulation, no toxin production, fast growth in a mineral medium (MM) and cellulose utilization (if possible) (Figure 1). Ideally, the strain should also have a wider temperature and pH window for fast growth. With these properties available, it is not so difficult to construct other desirable properties for a low cost PHA production such as high PHA accumulation level (over 90% cell dry weight), starch or even mixed substrate utilization, a high substrate to PHA conversion efficiency (over 50% g/g), and ability for production of short-chain-length (scl) and/or medium-chain-length PHA. For convenient downstream PHA recovery and purification, the above super strain should also have a large cell size, fragile cell wall, easy or inducible flocculation, etc. With many knowledge available today, it is a matter of time that the ideal and super PHA production is available for low cost PHA production (Figure 1 and Table 2). Further genetic manipulation allows the development of additional advantages for mimicking the chemical processing (Table 2). www.sciencedirect.com
Polyhydroxyalkanoates, challenges and opportunities Wang, Yin and Chen 61
Figure 1
Easy flocculation Fas t In M growth Mm edia
Hi acc gh PH um A ula tion
Growth at high or low pH Large size
No phage
Growth scl-and mcl producer at high or low temp
Cellulose utilizer
Fragile cell wall
No toxin
Clear genomic background Growth in mixed carbon sources High substrate to PHA conversion
No pathogen Easy genomic manipulation Starch utilizer
Current Opinion in Biotechnology
Desirable properties for a PHA industrial production strain.
To achieve a high final product concentration similar to chemical processing (Table 1), cell density should be reached as high as possible (>200 g/L), PHA content should be as high as over 90% or even to 95% of the cell dryweight (CDW) (Table 2). These goals can be realized by manipulating genes related to quorum sensing, oxygen
uptake and PHA synthesis mechanisms [24]. Since oxygen is always a limiting factor for high cell density growth, a separation of cell growth followed by an oxygen limitation induced PHA accumulation stage will be an ideal situation to maximize the PHA growth accompanied by maximal PHA yield. This separation on growth and
Table 2 Technology to be developed to lower PHA production cost Technology
Reasons and/or purpose
High cell density fermentation
Achieve effective growth and cells recovery
Growth cells in low cost substrates or mixed substrates Fast growing cells
Substrates contributed to over 60% of PHA cost
Fast growing CO2 utilizing bacteria able to produce PHA Open (unsterile) and continuous fermentation process PHA synthesis induced by oxygen limitation Ultrahigh PHA accumulation (over 95% PHA in cell dry weight) Increase substrate (mostly carbon sources) to PHA conversion efficiency Enlarging the PHA production cells Inducible cell flocculation Inducible cell lysis Cell disruption by PHA hyperproduction Extracellular PHA production
Reduce fermentation duration and avoid microbial contamination CO2 is a free substrate To save sterilization energy, reduce fermentation complexity and improve process effectiveness Oxygen is a limited factor in all high cell density growth To avoid expensive and complicated downstream PHA purification process Substrates contributed to over 60% of PHA cost
To allow more cellular space for PHA accumulation, this also allows easy cells recovery Allow easy biomass recovery after fermentation Allow easy PHA granules recovery after biomass harvest Save the biomass harvest process
Large PHA granules
Not limited by a small cellular space, also for easy PHA granule recovery Allow easy recovery of PHA granules from lysis broth
A synthetic cell combining the above properties
Achieve up-stream and down-stream competitivenesses
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Methodology Manipulation on quorum sensing and cell oxygen uptake mechanisms Screening targeted substrates utilizing bacteria able to produce high content PHA Minimizing bacterial genome, changing cell growth patterns Manipulating the CO2 uptake mechanism such as carboxysomes, etc. Screening for PHA producers able to grow fast in extreme environments such as high or low pH and temperature, high osmotic pressure, etc. Place PHA synthesis operons behind microaerobic promotor Manipulating the PHA synthesis mechanism and PHA synthases Removing pathways that consume substrates for non-PHA metabolisms, and/or reinforce PHA synthesis flux Engineering the cell division patterns and/or cytoskeletons Inducible expression of surface displaying adhesive proteins Inducible expression of cell lysis proteins Manipulating the PHA synthesis mechanism and PHA synthases Need new PHA synthesis mechanisms Manipulating the formation of PHA granules associated proteins An artificial cell with assembled functional DNA
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PHA production can be materialized by placing PHA synthesis operons behind microaerobic promotor [25]. Since substrates play the most important part in PHA cost, it is crucial to use low cost substrates such as mixed substrates (kitchen wastes), low cost starch, cellulose (and/or its hydrolyzed products) or even CO2. It is normally easier to isolate mixed substrates utilizing microorganisms than engineering them. However, it has become possible to manipulate the CO2 uptake mechanism such as carboxysomes, etc. to take up CO2 as substrate for cell growth [26,27]. Also, a combination of deletions on pathways that consume PHA monomers and over-expressions of PHA production pathways can lead to increase substrates to PHA conversion efficiency (Table 2) [28]. Development of competitive PHA production processes
Downstream processing of biological products is also costly. For example, it requires continuous centrifugation process to separate PHA containing cells from the fermentation broth [1]. While the cells are small and light with lower concentration in the broth, separation via continuous centrifugation is a long and energy intensive process. It is therefore important to enlarge the cell sizes, and/or construct inducible cell flocculation to ease the precipitation process. Systems and synthetic biology allow creation of pathways in PHA production bacteria combining cell division and surface display of adhesive proteins (Table 2). After cells separation from the broth, inducible cell lysis is important to release the intracellular PHA granules [29], this can be achieved by induced expression of lysis proteins such as lysozymes, exonucleases and proteinases, etc. Ideally, PHA synthesis mechanism should be exploited so that ultrahigh PHA production up to 95% in CDW can be reached. Such a high PHA accumulation level can lead to cell ruptures, easing the cell lysis process. PHA granules should be large in size for easy separation. By manipulating the PHA granule associated proteins, it is possible to increase PHA granule size [30]. Extracellular production of PHA will be an ideal way of making PHA since extracellular PHA accumulation is not limited by the small cell volume and is convenient for downstream extraction [31]. This points to a future with unlimited production of PHA via extracellular mechanism. For example, bacterial cell walls can be weakened by deleting some cell wall synthesis related genes. According to some related studies recently conducted in this lab, PHA could be produced extracellularly via weakened cell walls for the benefit of enhanced PHA production and extraction. The above cell properties including large cell size, ultrahigh PHA accumulation, inducible flocculation and lysis, large PHA granules, extracellular PHA synthesis and high cell density growth can be achieved by genetic Current Opinion in Biotechnology 2014, 30:59–65
engineering, pathway engineering and synthetic biology. There is still one setback of the current PHA production process, which is its discontinuity requiring energy intensive sterilization using high-pressure hot steam. To overcome this difficulty, PHA should mimic chemical processing using continuous and unsterile processes. Recently, it has become possible to recruit mixed cultures for continuous and unsterile production of PHA [32]. However, pure culture is preferred as it usually lead to more reproducible results. Fortunately, halophilic bacteria offer such possibilities as they grow in medium to high salt concentrations under high pH in a wide temperature window, such growth conditions can effectively prevent contamination by other microorganisms [33]. In the authors’ lab, we are able to grow halophilic bacteria in unsterile and continuous process in seawater like medium for at least one month without any microbial contamination [33], the halophilic bacteria have been proven easy for genetic manipulation, allowing the construction of above mentioned super PHA production strains [34]. Since the halophilic strains can grow in seawater in a continuous and unsterile way, the development of these types of strains for PHA industrial production can save a lot of fresh water and energy, reduce process complexity and thus, increase the competitiveness of PHA. High value added PHA production and applications
PHA that have been produced in large scale including poly(3-hydroxybutyrate) (PHB) [15], poly(4-hydroxybutyrate) (P4HB) [35,36], copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) [1], copolymers of 3hydroxybutyrate and 3-hydroxyvalerate (PHBV) [1], as well as similar copolymers of 3-hydroxybutyrate and 3hydroxyhexanoate (PHBHHx) [1]. Except the application as biomedical implant materials, all the above PHA are mainly developed as environmentally friendly biodegradable packaging materials which are low value added. To exploit the low value added yet expensive PHA materials produced in large scale, it is important that the applications can generate high value and quantity demand is large. Recently, high quality textile from PHBV has been successfully manufactured (Table 3). If the marketing is successful, this will open a large quantity market for PHA. On the other hand, it was reported that ultrahigh molecular weight PHA can be used to make ultrastrong fibers for fishing lines and fishing net, etc. [37,38]. The ultra strong property is also a good sale for high value. Recently, the authors’ lab succeeded in engineering the b-oxidation pathway encoded on the chromosomes of Pseudomonas putida and Pseudomonas entomophiles, allowing the synthesis of controllable PHA microstructures including formation of PHA homopolymers, composition www.sciencedirect.com
Polyhydroxyalkanoates, challenges and opportunities Wang, Yin and Chen 63
Table 3 Technology to be developed to increase PHA values Technology Controllable PHA synthesis with defined structures
Controllable formations of functional PHA
Increase PHA diversity Ultrahigh molecular weight Production of single chiral PHA monomers
PHA as medical implant materials PHA as good quality textiles
Reasons and/or purpose To form PHA homopolymers, defined ratios monomers in random copolymers and block copolymers To form PHA smart materials with properties of pH, temp, and wettability sensitivities or shape memory, etc. To find other possible applications To obtain ultrastrong PHA fibers Mostly as drugs or as lead compounds for drug molecule synthesis PHA are biodegradable and biocompatible polyesters PHA can be turned into textiles that have values higher than packaging materials
adjustable random copolymers and block copolymers [10,39,40]. Since PHA structures can be designed, we can obtain required material properties based on precisely designed and synthesized PHA. Especially on the block copolymers reported to have anti-aging property [41], a series of diblock copolymers including P3HB-b-P4HB [42], P3HB-b-P3HHx [43], and P3HP-b-P4HB [8] were successfully synthesized and found to have one or two improved properties over their two relative homopolymers, random copolymers or blend polymers. Most importantly, block ratios in copolymers can be adjusted, leading to adjustable polymer properties. Since functional groups can be inserted into PHA chains, such as double or triple bonds, epoxy, carbonyl, cyano, phenyl and halogen [44], graft PHA polymers can be synthesized by chemical modification of the PHA side chains, leading to dramatic changes in PHA properties. There are limitless possibilities to create new graft PHA homopolymers or copolymers. Finally, PHA monomers are mostly chiral hydroxyalkanoic acids (HA), microbial production of chiral HA can be achieved by deletions of partial b-oxidation pathway and PHA synthase phaC operon combined with over-expression of thioesterase genes in Pseudomonas entomophila [45], the resulting recombinant P. entomophila was able to produce various chiral HA, respectively, from their related carbon sources. Since chiral HA are important chemicals used as precursors or intermediates for the synthesis of various fine compounds including pharmaceuticals, antibiotics, food additive, fragrances, and vitamins [3,5], they have high value added applications waiting to be exploited. Future prospects
The recent advances on systems and synthetic biology will allow the design and construction of a PHA super www.sciencedirect.com
Methodology Engineering the PHA synthesis pathways, especially the b-oxidation pathways Additions of functional PHA as monomer precursors
Chemical grafting of PHA functional side chains Engineering the PHA synthase or operon Deleting PHA synthases in PHA synthesis pathway PHA can be turned into any shape for implant purposes Controllable crystallization of PHA fibers
production strain that will be able to grow fast to high cell density (>200 g/L) utilizing low cost substrates including cellulose, starch or even kitchen wastes under a very high carbon source to PHA conversion efficiency of at least 50% (g/g). The synthetic cell will be able to achieve oxygen limitation induced >90% PHA accumulation in cell dry weights. After completing the PHA production, the very large cells will be induced for flocculation precipitation followed by induced cell lysis to release large PHA granules. The PHA fermentation process will be conducted under unsterile and continuous way using seawater to save energy and fresh water, reduce process complexity so that the resulting PHA can be competitive in production cost with petroleum based plastics. The above super PHA production strain will be constructed to allow precisely control their PHA structures to form homopolymers, random copolymers and block copolymers as well as functional polymers with precise monomer structures and ratios for consistent properties. High value added applications based on unique PHA polymer properties and chiral hydroxyalkanoic acids will be developed for the high end markets.
Acknowledgements This project has been supported by State 973 Basic Science Research Project (Grant No.: 2012CB725201) and Natural Science Foundation of China (Grant No. 31270146).
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An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. This process opens a new area for reducing the cost in polyhydroxyalkanoates production.
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Current Opinion in Biotechnology 2014, 30:59–65
