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Microbial Production of Bioplastics--Polyhydroxyalkanoates
GUO-QIANG CHEN Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China
Living organisms are able to synthesize various biopolymers including DNA, RNA, proteins and polysaccharides. Beside these biopolymers, microorganisms, especially bacteria, are capable of synthesizing a family of polyesters, namely polyhydroxyalkanoates, abbreviated as PHA, as their intracellular carbon source and energy storage compounds. Worldwide many efforts have been made to produce these biopolyesters. Research has also been conducted to find high value added applications for this type of unique material.
1. INTRODUCTION
Polyhydroxyalkanoates (PHA) have shown extensive structure variety1. Depending on growth substrates and types of organisms used, the site chain R can change from a simple methyl group to functional structures containing unsaturated double or triple bonds, benzyl, halogens, cyanide or epoxy groups (Fig. 1)2. Both the monomer structures and monomer contents affect PHA physical properties. PHA can be very brittle, such as polyhydroxybutyrate (PHB) produced by many bacteria; it can also be flexible, such as copolyesters PHBV consisting of 3-hydroxybutyrate (HB) and 3-hydroxyhexanoate (HHx), and copolyesters PHBHHx consisting of HB and 3-hydroxyvalerate (HV). PHA can be elastic too when their monomers are 3-hydroxyoctanoate or 3-hydroxydecanoate (Table 1)3.
Figure. 1 General molecular structure of polyhydroxyalkanoates m = 1, 2, 3, yet m = 1 is most common, n can range from 100 to several thousands. R is variable. When m = 1, R = CH3, the monomer structure is 3-hydroxybutyrate, while m = 1 and R = C3H7, it is a 3-hydroxyhexanoate monomer. If R≥C3H7, the PHA are called medium-chain-length PHA; If R< C3H7, the PHA are referred to as short-chain-length PHA.
There are many bacteria capable of producing PHA. Among PHA, PHB is most commonly found in many bacteria. In order to find bacteria able to synthesize non-PHB polyesters, screening process will have to carry out. Although many PHA have been found, only three of them were produced in large scale for commercial exploitation, these are PHB4, PHBV5 and PHBHHx6. There are still a lot of unknown for the production of these unique polyesters. The cost for the production of PHA is still too high for application for biodegradable packaging. High value added applications, especially biomedical application and fine chemical application, may be realistic for the current PHA applications. Many efforts have been made in this area.
Table 1. Physical properties of various PHA in comparison with conventional plastics3
HB: 3-hydroxybutyrate; HV: 3-hydroxyvalerate; HHx: 3-hydroxyhexanoate
Figure. 2 Granules of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) produced by Aeromonas hydrophila. 20×1000 amplification
2. PRODUCTION OF POLYHYDROXYALKANOATES (PHA)
To exploit the application of PHA, large quantity of PHA has to be supplied. Although 90 PHA with various monomer units were reported in 1991 and this number is still increasing, application research on PHA can only be conducted with a handful of PHA that can be produced in sufficient quantity. The high cost associated with finding the right organism and developing an industrial PHA production process has contributed to the slow development of PHA production technology, this further leads to the high production cost for PHA. Thus, large scale application of PHA as environmentally friendly materials has been discouraged partially by the high cost. On the other hand, mechanical properties of PHA play an important role in their applications. Many approaches have been adopted to improve the flexibility of this unique material.
Screening for Industrial PHA Producing Microorganisms
Many bacteria are able to produce PHA, especially PHB1,7,8,9. However, there are very few of them that can be used for industrial production purposes. As an industrial PHA production strain, the microorganism should satisfy following requirements: rapid growth in cheap carbon sources, high PHA accumulation in the cells, high substrate to product transformation efficiency, harmless to animals, human and the environments, large in size for separation purposes, and easy lysis for PHA extraction. Ideally, the bacteria should also have a low oxygen demand so that cells can be grown to high density without encountering oxygen limitation, at the same time, the cells should also produce PHA with high molecular weight for application purposes. Due to the difficulty to isolate bacteria that satisfy so many requirements, only Alcaligenes latus10,11 and Rastonia eutropha5 (Formerly called Alcaligenes eutrophus) were used for PHB and PHBV industrial production. Other bacteria, such as Pseudomonas oleovorans and Aeromonas hydrophila which produce medium chain length PHA and copolyesters PHBHHx, respectively, do not meet all the above requirements12,6. However, they are still used for industrial production due to the lack of alternative strains. Therefore, it is extremely important to develop a rapid screening method that will allow the discovery of a suitable industrial PHA production strain from many bacterial strains available. Normally, the screening process of a PHA producing strain can be divided into the following steps: isolation and purification of a single clone, followed by growing the strain in shake flasks; centrifugation to collect biomass after cell growth, followed by freeze dry the cells; PHA extraction out of the cells; gas chromatographic study of PHA monomer structures; GC-MS (Gas chromatography and mass spectrascopy) study to confirm the PHA monomer structures. The entire process can last months before the ability of PHA production and the types of PHA produced by certain bacteria grown on certain substrate are known. This approach is labor intensive and time consuming. Although it has had a lot of disadvantages, the method has been practiced by many researchers worldwide. Obviously, this lengthy screening process will not be effective for screening large number of PHA producing microorganisms. At least two methods for rapid screening of PHA producing strains were developed. One is the Nile red method, another is the FT-IR approach. Using the Nile red method, all PHA polyesters show a similar fluorescence behavior, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. The examination of native PHB granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 mu g Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the PHB concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial PHA13. This Nile red staining method was successfully applied to distinguish PHA producing strains from non-PHA producers from many clones grown on Petri dishes14. However, the Nile red staining approach can not tell the PHA structures. If one observes the bacterial clones stained with Nile red on the Petri dish, it will not be possible to estimate the PHA contents and the types of PHA synthesized. Therefore, an improved method needs to be developed to overcome these setbacks of the Nile red staining method. A FT-IR method was shortly developed15. The method is rapid, convenient, non-invasive, combined with the possibility to distinguish short-chain-length and medium-chain-length PHA, as well as quantitatively assay the intracellular PHA contents16. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mcl HA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, show their strong characteristic band at 1728 cm-1, 1740 cm-1 or 1732 cm-1 respectively. Other accompanying bands near 1280 cm-1 and 1165 cm-1 help identify the types of PHA. The intensity of the methylene band near 2925 cm-1 provides additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm-1, 1744 cm-1 or 1739 cm-1, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. Therefore, by scanning the bacterial cells, it will be possible to tell the approximate contents of PHA in the cells, or to tell the types of PHA synthesized by the cells within 10 seconds. Thus, the FT-IR technique will allow the rapid screening of PHA producing strains from among large number of bacterial colonies. A broad screening process using the FT-IR technique was carried out17. Samples were collected from various geological locations around China. The FT-IR method proved very effective. It was found that PHA compositions depend very much on the geological locations. In some locations, bacteria mainly synthesized short-chain-length PHA, in other locations, medium-chain-length PHA (mcl PHA) was accumulated by inhabiting bacteria. Additionally, the synthesis of blend polymers consisting of PHB, short-chain-length PHA and mcl PHA is a common phenomenon among the bacteria studied. 40% of the 371 strains cultivated on six substrates were able to synthesize PHA, with many of them making blends of PHB and mcl PHA. This result will help polymer researchers to identify sources of PHA synthesizing bacteria.
Production of Polyhydroxybutyrate (PHB)
2.2.1 PHB Production by Bacillus spp. Bacillus spp. were among the very first to be reported as PHB producers18. However, we were surprised to learn that no PHB production research in terms of process development was conducted with this organism although Bacillus spp. have long been known to grow rapidly, they are also capable of using various cheap carbon sources for growth, in addition, they are very resistant to contamination by other bacteria. Chen et al reported the production of PHB from 11 Bacillus spp. randomly selected from German Culture Collection (DSM) never exceeded 50% when growth was conducted in shake flasks8. To investigate the possibility for PHB production using Bacillus spp., a Bacillus strain isolated from molasses contaminated soil was used as a model19. It appeared that PHB formation was growth associated, factors that normally promote PHB production including high ratios of carbon to nitrogen, carbon to phosphorus and low oxygen supply, did not lead to high PHB production. Instead, these factors resulted in sporulation, which further leads to reduced PHB contents and cell dry weight. Molecular weights of PHB produced by this Bacillus sp. were all low. The competition of PHB synthesis and sporulation seemed to be the reason for low PHB production. Therefore, Bacillus spp. may not be a suitable PHB industrial production strain. Furthermore, the thick Gram positive cell wall will make the breakage of cells and PHB extraction difficult.
2.2.2 PHB Production by Alcaligenes latus Alcaligenes latus is one of the strains that satisfy the requirement for industrial PHB production20. The strain grows rapidly in sucrose, glucose and molasses. PHB accumulation can be as high as over 90% of the cell dry weight21. Chemie Linz AG/Austria (later btf Austria) produced PHB in a quantity of 1000 kg/week in a 15 m3 fermentor using Alcaligenes latus DSM 11244. The cells were grown in mineral medium containing sucrose as carbon source. The PHB produced by Alcaligenes latus has been used to make sample cups, bottles, syringes for application trials. The PHB production and processing technology are now owned by Biomer in Germany. Different products including combs, pens, bullets have been made from PHB produced by Alcaligenes latus.
2.2.3 PHB Production by Ralstonia eutropha Ralstonia eutropha was used to conduct PHB production research in a 1m3 fermentor under the joint action of Institute of Microbiology affiliated to the Chinese Academy of Sciences and Tianjin Northern Food Inc./China. Growth was carried out for 48 h in glucose mineral medium. At the end of the cell growth, cell density reached 160 g/l. The cells produced 80% PHB in their dry weight. Most surprisingly, the strain grown to such a high density did not require oxygen enriched air. This was perhaps the highest cell density for PHB production achieved in pilot scale production.
2.2.4 PHB Production by Azotobacter vinelandii Azotobacter vinelandii strain UWD was demonstrated to grow rapidly in molasses medium22. The strain has a large size, ranging from 1 to 8 mm. It can produce PHB up to 90% of cell dry weight. At the same time, the strain produces PHB with a molecular weight ranging from 1 to 4 million Dalton23, this is rarely seen with any microorganism. PHB production could be promoted by lower aeration, therefore, PHB production can be separated into two-stage: one for cell growth under high aeration and another for PHB accumulation under lower aeration24. In a small scale lab top fermentor, 36 g/l PHB were produced from molasses after 48 h of growth. A collaboration between the Microbiology Lab at Tsinghua University and Guangdong Jiangmen Center for Biotech Development/China for pilot PHB production by A. vinelandii UWD was carried out on molasses medium. The pilot study was done in a 4 m3 fermentor without automatic oxygen supply control. After 48 h of growth, the cells reached a density of 75-80 g/l. The PHB content in the cells was as high as 72% of the cell dry weight. The cell size was at least 6 mm in diameter. Due to the high PHB accumulation efficiency and the large cell size, separation of biomass from the fermentation broth using continuous disk centrifuges was convenient. At the same time, the cells were easily broken by a 0.2% SDS solution at 60oC for 2 h, making the downstream processing relative easy. Major problem with this strain has been the difficulty to grow the cells to high density, as this strain requires high dissolved oxygen concentration for high density growth. The supply of oxygen enriched air for industrial fermentation is impossible due to its explosive danger and high cost of pure oxygen supply. PHB produced by the strain is now under study by the Institute of Polymer Sciences and Engineering at Tsinghua University. Major efforts have been focused on improving the mechanical strength and the exploitation of tissue engineering application for this polyester.
Production of Copolyesters Consisting of 3-Hydroxybutyrate and 3-Hydroxyvalerate (PHBV)
ICI Bioproducts & Fine Chemicals (now Zeneca) was the first to really produce PHA in large scale, namely, copolyesters (PHBV) of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV)5. The production strain is Ralstonia eutropha, the strain is able to grow on glucose and produce the copolymer PHBV to a density as high as 70-80 g/l after over 70 h of growth. Shampoo bottles were produced from PHBV (Trademarked as BIOPOL) and were available in supermarkets in Europe. However, due to the economic reason, the Biopol products did not succeed and the PHBV patents were sold to Monsonto. Hangzhou Glutamate Ltd./China, in collaboration with the Institute of Microbiology affiliated to the Chinese Academy of Sciences, has developed a model process that can produced PHBV in high efficiency. Without supply of pure oxygen, R. eutropha grew to a density of 160 g/l cell dry weight within 48 h in a 1000 L fermentor. The cells accumulated 80% of PHBV with a production efficiency of 2.5 g/h/l. The HV content in the copolymer ranged from 8-10%. This process can significantly reduce the production cost for PHBV. Only by achieving the high growth rate, high PHBV production efficiency and high cell and PHBV densities can the polymers become economically competitive. We assume that PHBV or other PHA can become cost effective after extensive improvement in fermentation process and downstream process.
Production of Copolyesters Consisting of 3-Hydroxybutyrate and 3-Hydroxyhexanoate (PHBHHx)
Recently, Tsinghua University in Beijing/China, in collaboration with Guangdong Jiangmen Center for Biotech development/China, KAIST/Korea and Procter & Gamble in USA has succeeded in producing PHBHHx by Aeromonas hydrophila grown in a 20 cubic meter fermentor6. The PHBHHx production was carried out on glucose and lauric acid for about 60 h. Cell dry weight reached 50 g/l, only 50% of PHBHHx was produced in the cell dry weight. The extraction of PHBHHx was a very complicated process involving the use of ethyl acetate and hexane, which increased the polymer production cost dramatically. PHBHHx produced by Jiangmen/China is now been exploited for application in areas of flushable, nonwovens, binders, films, flexible packaging, thermoformed articles, coated paper, synthesis paper, coating systems and medical devices (www.nodax.com). Copolymers consisting of HB and medium-chain-length HA have been trademarked by P&G as NODAX. Current production cost for PHBHHx is still too high for real commercial application. However, many efforts have been made to improve the production process for PHBHHx including the downstream process technology.. Most efforts have been focused on increase cell density and simplify the downstream process. A better production strain able to utilize glucose will be one of the most important issues of reducing the PHBHHx production costs.
Production of Copolyesters Consisting of Medium-Chain-Length Hydroxyalkanoates
Medium-chain-length (mcl) PHA can be produced by Pseudomonas oleovorans and Pseudomonas stutzeri as well as other Pseudomonas spp. It was reported that mcl PHA can be produced at costs below US$10/kg if production scale is 1000 tones/year using the P. oleovorans grown on octane. However, mcl PHA made up only less than 40% of the cell dry weight. It would be very important if a strain can produce at least over 50% mcl PHA. Strain P. stutzeri 1317 isolated from oil contaminated soil was found to grow on a variety of carbon source including glucose and soybean oil26. The strain produced over 63% mcl PHA when grown on soybean oil. While on glucose, 51% mcl PHA was synthesized by this organism. The strain is currently under intensive investigation on the possibility to increase the mcl PHA production level.
3. APPLICATION OF POLYHYDROXYALKANOATES AS BIOMATERIALS FOR TISSUE ENGINEERING
Applications for PHA can be found in areas of flushable, nonwovens, binders, films, flexible packaging, thermoformed articles, coated paper, synthesis paper, and coating systems (www.nodax.com). However, Current PHA production cost is still too high to satisfy such low value added demand. Therefore, we believe that high value added application should be more realistic. As PHA is biodegradable, and possibly biocompatible, its application as biomaterials or tissue engineering materials should be very attractive. To test the biocompatibility of PHA, three polymers were selected, namely, PHB, PHBHHx and Poly-L-lactic acid (PLA). Mouse fibroblast cell line L929 was inoculated on films made of PHB, PHBHHx and their blends, as well as PLA. Results showed that PHBHHx had the best biocompatibility, followed by PHBHHx/PHB blend and the least biocompatible polymer was PLA27,28. Since mechanical strength of PHBHHx is among the best compared with PHB and PLA, therefore, it is expected that PHBHHx possessing better biocompatibility and mechanical strength will have a promising future in tissue engineering application. To test this promise, polymer scaffold systems consisting of poly(hydroxybutyrate-co- hydroxyl- hexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture29. Blend polymers of PHBHHx/PHB were fabricated into three- dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited at appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days. This further showed that PHBHHx is a good candidate for tissue engineering application.
4. CONCLUSION
Microbial production of PHA has been developed over the past two decades. PHB and PHBV fermentation technology has been very well exploited. Production costs for these two types of polymers have been significantly reduced. Better performed polymers, especially copolyesters consisting of HB and mcl HA, should be the focused point for development. However, current industrial production strains for this type of polymers suffer from slow growth, low substrate to product transformation efficiency, dependent on expensive substrates. Efforts should be made to isolate or construct high productive strains and to improve fermentation and downstream processing technology. Before the PHA production cost can be brought to the point where it can compete with conventional plastics, high value added application in medical fields should be the real destination for PHA. As these polymers possess not only better mechanical properties and processibility but also better biocompatibility, therefore, PHA application as tissue engineering materials looks very promising.
ACKNOWLEDGEMENTS
The Natural Science Foundation of China Grant No. 30170017 and 20074020, Tsinghua University 985 project, State 9th Five-Year R&D Project, and Procter & Gamble provided financial supports for these works.
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