Introduction to PHA
Polyhydroxyalkanoate (PHA) is one of storage polyesters synthesized in some bacterial cells under unbalanced nutrient conditions (e.g. short of nitrogen, or phosphorus, or magnesium), functions as starch in plants and fat in animals. It has been found to occur in over 300 genera of bacteria, encompassing Gram-positive and Gram-negative species. Over 90 kinds of PHA have been characterized, the majority of them are composed of D-(-)-poly-3-hydroxyalknoic acid monomers ranging from C3 to C14 (figure 1). With different R groups, chain lengths, and site of hydroxyl group in monomers, various PHAs are formed.
PHA can be classified into three groups: short-chain-length PHA with C3-C5 monomers, such as poly-3-hydrobutyrate (PHB); medium-chain-length PHA with C6-C14 monomers, such as poly-3-hydroxyoctanoate(PHO); and copolymer by different monomers, such as poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV).
The water insoluble PHA exhibits high molecular weight, thermoplastic and/or elastomeric features as well as other interesting physical and material properties. Therefore, they have attracted increasing attention from scientific and industrial communities due to their interesting properties including biodegradability, biocompatibility and piezoelectricity.
Manufacturing technology of PHA
BTL have incorporation fulfilled the breakthrough tasks of biodegradable plastics, which includes four sub-subjects: production of polyhydroxybutyrate (PHB) using waste molasses as substrate; production of PHB by recombinant microorganism; production of PHB and its copolymer PHBV using hydrolyzed starch as substrate; PHB property modification and its application research. Besides, the third generation of PHA namely PHBHHx has been produced in a certain scale for the first time in the world providing broad prospects for the research on biodegradable plastics commercialization in China.
Production of PHA using waste molasses as substrate:
In this sub-subject, the major task is pilot-scale study on PHA (The typical representatives is polyhydroxybutyrate PHB) manufactured by fermentation using waste molasses as substrate which helps to design proper process route and provides a basis for industrial production. The technical and economic indexes were: 70g/L cell dry weight, 70% PHB yield, millions molecular weight, more than 95% product purity and less than US$8/Kg the production cost of PHA. BTL resolved the following key technical points by doing a great deal of work:
(1) reduced the cost of fermentation raw material by using waste molasses as substrate.
(2) Azotobater Vinelandii UWD was used as the producing strains in the fermentation production, which not only can synthesize high molecular weight PHB, but also can utilize waste molasses.
(3) solved the problem of supplying oxygen under high density culture.
(4) simplified the process of breaking cell and releasing intracellular PHB particles.Finally, the cost of PHB was successfully reduced to US$6/Kg.
Production of PHA by recombinant microorganism
In this sub-subject, the main task were constructing gene engineering recombinant strains to carry out the laboratory research on PHB fermentation production and establishing a new process using cheap carbon source(starch hydrolysate) to ferment, which helps to provides a basis for pilot-scale and industrial production. The assessment target and technical&economic indexes were: 200g/L cell dry weight, 70% PHB yield, more than 95% product purity, less than US$8/Kg the production cost of PHA.
The following key technical points were solved:
(1) constructed multifunctional new strain of gene engineering which can efficiently express and produce PHB and synthesize biodegradable plastics utilizing cheap carbon source (starch hydrolysate) to reduce the cost of fermentation raw material; solved the problem of supplying oxygen under high density fermentation; developed the process simplifying breaking cell and releasing intracellular PHB particles; adopted a new purifying process not using organic solvent extraction.
(2) did research on optimization controlling condition of fermentation technology and fermentation process using multifunctional strain so as to implementation requirements of fermentation in the tackling technical indexes.
(3) researched the process of cell post-treatment and PHB purification whose cost was as low as possible.
The optimal strain VGI (pTU14) was chose from more than 40 constructed strains. The experiment results indicated that the content of PHB accumulated in the strain cells reached to more than 90%. More than 90% cells had decomposed when the fermentation finished because this strain had cell decomposition gene. When VGI was adopted, the strain content (dry weight) in the fermentation liquid was 215.9 g/L, PHB accounted for 89.7% of the strain dry weight, the product purity was over 98% and the cost of PHB was US$6 /Kg.
Production of PHA using hydrolyzed starch as substrate
In this sub-subject, the main task were carrying out pilot-scale study on PHA production by fermentation based on experiments and establishing a new process using cheap carbon source(starch hydrolysate) to ferment but not using organic solvent extraction to purify. The task demanded more than 130g/L strain content in the fermentation liquid, 1.5g(PHA)/(L•h) production intensity and less than US$7 /Kg production cost.
In the 1.2t fermentor, high density culture of cells was realized by effectively controlling the equilibrium growth condition of cell growth and non equilibrium growth condition of PHA accumulation. In the five batches experiments, the average fermentation time was 58h, the average cell content was 134.3 g/L(dry weight) and the maximum reached to 164.4 g/L(dry weight), PHA accounted for 78% of the strain dry weight and the maximum reached to 80%, the average production intensity was 1.84g(PHA)/(L•h) and the maximum reached to 2.18g(PHA)/(L•h), the average conversion rate of glucose to PHA was 0.28 the maximum reached to 0.30.
Moreover, not only the extraction process was simplified and the extraction efficiency was improved but also the production was reduced through collecting PHA particles by centrifuge after cell decomposition. The average extraction rate of PHA was 84.3, purity was over 95% and the cost of PHA pilot scale production was US$5-6/Kg.
PHB property modification and its application research
In order to be plastic product, PHA manufactured by microbial fermentation must be modified. The main task of this sub-subject were producing fine performance degradable polymer materials by combining various additives(such as plasticizer, foamer fluidity modifier, etc.) with other polymer materials and investigated their application in the field of thin film, vessel, medicine, agriculture so that their application performance were determined furtherly. Meanwhile, the research on reducing cost was done.
The assessment target and technical&economic indexes were: doing some research on PHA process technology in order to obtain fine performance(process and physical properties) degradable polymer materials; developing and researching on the application of various PHA products. The following work was done:
(1) modification of PHB
Researched PHB crystallization behavior under different conditions, especially investigated PHB crystallization process and the influence of one-component and two-component nucleating agents(containing NH4Cl,BN, CaCO3 and various aliphatic dibasic acids) on PHB crystallization; studied blending modification of PHB; studied the synergetic effect between plasticizer epoxy soybean oil and nucleating agents TMB-3, TMB-4, TMB-5 and Tween 80. Finally, the extrusion and injection process of modified PHB were implemented successfully and the samples were obtained.
(2) application of PHB
Made micro balls and micro rods by using PHB、bovine serum albumin(BSA) and medicines such as D-18 methyl levorotation norethisterone(LNG) and studied their drug release rate furtherly; researched the preparation process and the biocompatibility、in vitro degradation and surface modification of PHB stents; carried out Osteoblast implantation and culture experiments on PHB three-dimensional porous stents corporating with Academy of Military Medical Sciences; researched surface coating of PHB on biodegradable dishware(rice bowl), in which not percolated dishware in boiling-solution was made by brush coating on the dishware pressed from farm land straw.
Novelty
Fast Selected the strains which can produce the third generation biodegradable plastic by Fourier infrared nondestructive inspection technology invented by the researchers themselves.
Implemented the high density culture of strains in the process of the third generation biodegradable production and the new extraction process in the downstream of the methods of organic and inorganic.
Successfully developed the production process of the third generation biodegradable plastic PHA(that is PHBPHHx) in small scale, middle scale and large scale, in which the substrate was glucose, the cell dry weight was 50-60 g/L, the content of PHBHHx was 50-60% and the product purity was over 95%. It is the first success both in China and abroad.
For the first time, biodegradable butanedioic acid and epoxy soybean oil were added to the PHB as the plasticizer, which not only improved the mechanical properties of PHB but also had no influence on its degradability.
Benefit evaluation
It was reported that the PHB production cost in Britain was 17-22 dollar/Kg, however, the evaluated cost by BTL researcher was less than US$8/Kg. The direct economic benefit of the third generation biodegradable plastic PHBHHx was one million dollars.
Because of their diversity structure and new material properties result from structural change, PHAs are applied very widely. For example, they can be applied in the low additional value products such as biodegradable liquid packaging materials、food packaging materials、paper coating materials and sanitation materials, the high additional value products such as tissue engineering materials、electronic engineering materials and optical materials, some one-off articles such as high-grade packaging materials and composite materials、release carrier for drugs and pesticide、new medical materials(bone plates and bone nails) and so on, which can produce important economical、social and environmental value.
中文
