Study On The Production Of Polyhydroxyalkanoates By The Thermophilic Chelatococcus Daeguensis TAD1

Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microorganismsfrom renewable resources (such as carbohydrate and fatty acid) as intracellular carbon storagecompounds. The physical properties of PHAs are close to that of traditionalpetrochemical-based plastics. In addition, many PHAs have interesting properties, such asbiocompatible, piezoelectricity and optical activity, and have been recognized as one of thebest raw material for medical materials. With the exhaustion of petroleum resources, PHAshave attracted growing interest as suitable alternatives to petrochemical-based plastics.Thermophilic bacteria are of great interest for the production of PHA compared to that ofmesophilic bacteria, because of the numerous advantages on operating fermentation atelevated temperatures, which is of benefit for reducing the production cost of PHAs. Forinstance, the diffusion rates and solubility of chemicals increase at elevated temperatures,while the risk of contamination decrease significantly because of fewer microbial organismscan survive at elevated temperatures. Moreover, thermophilic fermentations are self-heatedsystems because the heat generated by the cell’s metabolism during high-cell-density (HCD)growth and also by stirring can be used for providing heat to the fermentation process itself.Thus, both heating and cooling costs are lowered. Additionally, the significance of enzymesderived from thermophiles has increased as they has turned out to be technologically valuable.In this context, the thermostability of PHA synthase might be a novel feature with a highpotential in biotechnological processes, such as in vitro PHA biosynthesis. In spite of theseadvantages, few PHA-accumulating thermophiles with have yet been found, and thesethermophiles have no potential for industrial production due to their extremely low growthactivities.In our group, we succeeded in isolating a thermophilic PHA-accumulating bacteria(Chelatococcus daeguensis TAD1) from the biotrickling filter system used for the removal ofnitrogen oxides. To the best of our knowledge, this is the first report on the production of PHAby the genus of Chelatococcus daeguensis. However, the PHA content of this bacterium(56.3%cell dry weight) is low. We therefore utilize Nile red agar plate to screen for theefficient TAD1S, which could effectively accumulate PHA, and the cytoplasmic PHAinclusions in TAD1Swere then investigated by staining the biopolymer with Nile red andobserving the cells under a fluorescence microscope. The architectural features and materialfeatures as well as content of PHA produced by TAD1Swere determined by gaschromatography, fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). This study then characterizes the PHA accumulation of C. daeguensis TAD1Sat elevatedtemperatures. After that, further investigation refer to the influence of temperature, molarratio of carbon to nitrogen, and carbon source on PHA accumulation were conducted to obtainthe optimal PHA amount by C. daeguensis TAD1S. Then, the effect of various factors on thePHA production by C. daeguensis TAD1Swas investigated to reduce the inhibition of highglycerol concentration, and a PHA fermentation system based on glycerol was thusestablished. Finally, with the aim of build up theoretical model for large scale production ofPHA, a5-l bioreactor was conducted to investigate the characterization of PHA production byC. daeguensis TAD1Sunder various fermentation technology.Under the optimal conditions (glucose20g/l,45oC,180rpm), the biomass and PHAcontent of C. daeguensis TAD1Sare2.71g/L and73.6%, respectively. The PHA wasdetermined by several analytic technology. The results showed that they are almost all poly(3-hydroxybutyrate)(PHB)(99%), indicating that C. daeguensis TAD1Spossess scl-PHAsynthetic pathway.The PHB accumulation of C. daeguensis TAD1Sis along with the cell growth duringlogarithmic phase, indicating that C. daeguensis TAD1Sis a growth-associatedPHA-accumulating bacteria without obvious nitrogen limitation. The suitable temperaturesfor PHB production by C. daeguensis TAD1Swere in the range from45oC to55oC. The PHAaccumulation occurred even if the temperature is up to60oC, indicating that the PHBbiosynthesis enzymes of C. daeguensis TAD1Sshould exhibit strong thermostability. Thenitrogen limitation could dramatically enhance the PHB accumulation. Meanwhile, eventhough the C/N was as low as5, C. daeguensis TAD1Sstill exhibited considerable PHBcontent (55.7%of CDW, w/w), showing strong tolerance to high concentrations of nitrogen.Under the optimal conditions (fermentation time28h, temperature50oC, C/N=30), thebiomass, PHB content and PHB amount of C. daeguensis TAD1Sare4.50g/L,76.5%and3.44g/L, respectively.C. daeguensis TAD1Swas able to utilize the sodium salts of short chain length (scl) fattyacid (C2-C6) for synthesizing PHB, indicating that the strain possesses the scl-PHAbiosynthesis pathway. C. daeguensis TAD1Sis obviously capable of accumulating PHBamounting to47.9%(w/w) of CDW from starch without pre-hydrolyzed. Studies focusing onthe PHA production from glycerol attracted more and more concerns. However, almost all ofthem operated at mesophilic environment (below40oC). In this study, we first report thatthermophilic C. daeguensis TAD1Sis able to accumulate PHB at elevated temperatures.Under the optimal conditions, the biomass, PHB content and PHB amount of C. daeguensis TAD1Swere8.12g/L,80.8%and6.56g/L, while the product yield and productivity were0.33g PHB/g glycerol and0.21g/L/h.With the aim of building up theoretical model for large scale production of PHA, a5-lbioreactor was conducted to investigate the characterization of PHA production by C.daeguensis TAD1Sunder various fermentation technology. Under two-stage fed-batchfermentation, the nitrogen limitation during last stage could significantly improve the PHBamount, and therefore of benefit for the downstream isolation and purification. The highestPHB content was obtained at40h, at this moment, the biomass, PHB amount, product yieldand productivity of C. daeguensis TAD1Swere25.4g/L,17.4g/L,0.26and0.434g/L/h,respectively.Furthermore, we succeeded in screening a new PHA-accumulating bacterium (K5) fromthe source of C. daeguensis TAD1S(a biotrickling filter used for the removal of NOx). ThePHA produced by K5was determined to be also poly (3-hydroxybutyrate)(PHB), indicatingthat C. daeguensis TAD1Spossess scl-PHA synthetic pathway. Meanwhile, the optimalfermentation temperature and initial pH are45oC and7.0, respectively.This work initially reveals the characterization of the PHA accumulation based onvarious carbon sources by thermophilic C. daeguensis TAD1Sand Bacillus shackletonii K5atelevated temperatures, which will play the theoretical foundation for later strain modification,process optimization of large-scale fermentation.