Screening And Optimizing Fermentation Conditions Of Polyhydroxyalkanoates-producing Bacterial Strains

Chemical synthesis plastics have brought great convenience to human life, but italso brought serious white pollution because it is undegradable. On the other hand, theraw material of chemical synthesis plastics—petroleum is increasingly in short supply.So research on the bio-degradable plastics is more important. Polyhydroxyalkanoates（PHA） has been considered as a good candidate to substitute petroleum derivedplastics. PHA, as a kind of polyesters of3–hydroxyalkanoic acids produced asintracellular granules, is synthesized and accumulated intracellularly under unfavorablegrowth conditions. It has showed potential applications in environment, biomedicinedomain, papermaking, electronic field, optical field and so on due to many advantages,such as biodegradable, non-toxic and environmental friendly.Recent research on PHA was focused on screening strains with high PHA yieldand developing PHA applications. In this study, soil or water samples were collectedfrom environment, and a polyhydroxyalkanoate-producing strain was isolated fromenvironment. This strain was identified and named Rhizobium sp. H2-5according to itsmorphological features, physiological and biochemical tests as well as16S rDNA genesequence analysis. The fermentation conditions and also the fermentation modes ofstrain H2-5were optimized and studied, which has improved the PHA content.The main research aspects and results are as below:1. Screening and identifying strains producing polyhydroxyalkanoates. Soil orwater samples were collected from environment, and a polyhydroxyalkanoate-producing strain was isolated from environment by enrichment culturing, Nile bluefluorescence staining, Sudan black staining, Fourier transform infrared spectraelemental analysis. This strain was identified and named Rhizobium sp. H2-5accordingto its morphological features, physiological and biochemical tests as well as16S rDNAgene sequence analysis.Morphological characteristics indicated that it was a kind of gram-negative rodbacterium lacking spore. Colonies on LB plate are circular, translucent and the surfaceis smooth with an entire edge.The color of the colony is light yellow. Biologyphysiological and biochemical tests indicated that strain H2-5can not utilize starch andcitric acid salt. V.P test, MR test and H2S producing test are all negative. Strain H2-5growing well on spore-producing medium but no spore produced. The growth curve of strain H2-5showed that strain H2-5has a short adaptive phase（3h）, and it has been instationary phase at18h after inoculation, so strain H2-5is short-adaptive andrapid-growing.2. Optimizing of fermentation conditions of Rhizobium sp. H2-5. The data ofsingle factor experiments showed that the best carbon source and nitrogen source wereglucose and peptone respectively. PHA content was affected greatly by pH,temperature, inoculation volume and C/N, so the orthogonal test was designed bySPSS software. The orthogonal test results showed the optimum fermentationconditions were as below: the initial pH7.0, fermentation temperature30℃,fermentation medium volume in each shake flask100mL/500mL, inoculation volume4%, C/N100/4. After optimization, PHA content was above50%, with a increase of20%—22%.3. Studying on the fermentation modes of strain Rhizobium sp. H2-5.12.5g/LNH4Cl was added at24h and39h respectively. The result showed that fed-batchfermentation can improve the CDW and PHA content obviously, which was not up tothe industrialized scale. Two-step cultivation, which first collecting the whole cellsgrowing in rich nitrogen medium for36h and then putting them into poor nitrogenmedium, can get the high yield of the CDW and PHA.