الفهرس 
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Abstract
Nowadays, plastics (Synthetic polymers) raised major concerns among scientists due to being non-biodegradable and produced from nonrenewable origin as petroleum. The search for substitutes became a must. Polyhydroxyalkanoates (PHA) polymers satisfied these needs and became an efficient rival for plastics in the market. However, its high production cost in comparison to plastics constrains its use. Accordingly, in the present study some strategies were tracked for decreasing the cost of production of PHA by A. baumannii isolate P39 (Genbank accession number: KC876036 ) , B. cereus isolate P83 (Genbank accession number: KC876035), and A. macrocytogenes isolate P173 (Genbank accession number: KC685000 ). These included: preliminary PHA production at large scale level using 14 L laboratory fermentor; studying different methods of polymer recovery and characterization of both the polymer and PHA synthase enzyme, key limiting enzyme in PHA biosynthetic pathway.
In this study, production of polyhydroxyalkanoates biopolymers by A. baumannii isolate P39, B. cereus isolate P83, and A. macrocytogenes isolate P173 was carried out in a 14 L laboratory fermentor based on the optimum shake flask conditions obtained from the previous study. Regarding A. baumannii isolate P39, after applying shake flask optimum conditions on 14L fermentor (28°C temp, 200 rpm agitation, 1 vvm aeration), the PHB production and biomass levels increased than shake flask, but the time needed to reach maximum PHB production shifted from 48 hours in shake flask to 72 hours on fermentor. By decreasing aeration level to 0.5 vvm, PHB production maximum peak was shifted from 72 hours of incubation to 24 hours. Moreover, the PHB percentage increased from 14% to 28% per dry weight of bacteria. Thus, the optimum fermentation conditions were 28°C temp, 200 rpm agitation, 0.5 vvm aeration, and initial pH 7.2. For B. cereus isolate P83, the optimum conditions of shake flask (28°C temp, 200 rpm agitation, 1 vvm aeration) were implemented in the fermentor where a 1.4 fold increase in PHB production in a shorter time than shake flask (24 rather than 48 hours) was achieved. By testing the effect of aeration on PHB production, it was found that the best aeration level was 2 vvm which led to increasing PHB percentage per dry weight from 49% using 1 vvm aeration to 53%. In brief, the best fermentation conditions were 28°C temp, 200 rpm agitation, 2 vvm aeration, and 7.2 initial pH. In case of A.macrocytogenes isolate P173, using the optimized conditions obtained on the shake flask (200 rpm agitation, incubation temperature 37°C, initial pH 7.2, and 5% v/v inoculum size) the level of PHB production decreased, and the time needed to reach the maximum level shifted from 24 hours to 48 hours. By increasing inoculum size to 10% v/v, PHB production increased about 2.4 fold, and its maximum peak was attained in 24 hours of incubation. Upon controlling pH to 7.2, PHB production decreased. After studying different aeration levels (0.5, 1, and 2 vvm), the highest PHB production was achieved using 1 vvm aeration. from the obtained results, the best conditions for PHB production using the respective isolate were 200 rpm agitation, incubation temperature 37°C, initial pH 7.2, and 10% v/v inoculum size.
To control the poly-β-hydroxybutyrate (PHB) biopolymer production by the tested isolates, kinetic Modelling of the fermentation process should be addressed. Logistic and Leudeking–Piret models were used for describing cell growth and PHB production, respectively. They showed good agreement with the experimental data describing both cell growth and PHB production (average regression coefficient r 2:0.999). The growth associated production of PHB biopolymer was confirmed using Leudeking–Piret model. Addition of acetate as victim substrate in the fermentation medium confirmed that PHB acts as an electron acceptor for both A. baumannii isolate P39, and A. macrocytogenes isolate P173.
Different methods (Digestion by sodium hypochlorite, dispersion of both chloroform and hypochlorite, mechanical dispersion and chloroform extraction) were used for polymer recovery. The choice of the best recovery method was based on PHB concentration recovered and its molecular weight. According to the obtained results, the most suitable method of recovery for A .baumanni isolate P39 was chemical digestion using sodium hypochlorite since it produced the highest molecular weight with promising PHB concentration. Regarding B. cereus isolate P83, the best method was extraction using chloroform, it assisted in producing high concentration of polymer with high molecular weight and low polydisperisty index. For A. macrocytogenes isolate P173, the highest polymer concentration and molecular weight with low polydisperisty was recovered using chemical digestion with sodium hypochlorite.
The PHA produced by the tested isolates was characterized using IR and 1H-NMR spectroscopy to configure its monomer composition. They showed that the PHA produced is a homopolymer of polyhydroxybutyrate. 1H-NMR showed that glycerol was a chain terminator of PHB produced using corn oil by either A. baumannii isolate P39 or B. cereus isolate P83. Gel permeation chromatography was used to measure average molecular weight of polymer produced by the respective isolates. The highest molecular weight was extracted from B. cereus isolate P83 (26900g/mole), while the lowest molecular weight was produced by A. baumannii isolate P39 (16000 g/mole).
The PHA synthase enzyme, key limiting enzyme of PHB biosynthesis, activity was measured by DTNB assay. The highest recorded activity was for A. baumnnii isolate P39 (600 U), and highest specific activity was for B. cereus isolate P83 (1500U/mg). In addition, the PHA synthase assay showed 2 min lag time for A. macrocytogenes isolate P173, 1 min for B. cereus isolate P83, and no lag time for A. baumnnii isolate P39.
Genetic analysis of PHA synthase enzyme of the tested isolates showed that this enzyme belongs to α/β hydrolase super family. Moreover, PHA synthase class III was found in A. baumannii isolate P39 and A. macrocytogenes isolate P173 while class IV is found in B. cereus isolate P83. The nucleotides sequences were submitted into the GenBank under the following accession codes: KX358864, KX358865, and KX358863 for PHA synthase of A. baumannii isolate P39, B. cereus isolate P83, and A. macrocytogenes isolate P173 gene, respectively.
Analysis of the putative tertiary structure of PHA synthase enzyme was carried out using MODAS class prediction software and Swiss model software. from the obtained results, the structural class of the three PHA synthase was estimated by MODAS software to be multidomain protein. Swiss model revealed the conserved cysteine residue in the known nucleophilic elbow of α/β hydrolase super family which is surrounded hydrophobic amino acids residues to act as a tunnel for entrance of substrate inside PHA synthase enzyme. Moreover, the absence of transmembrane helix in the isolated PHA sequences of the tested isolates was confirmed using TMHMM software. Taken together, the results of enzymological and molecular characterization of PHA synthase enzyme support the PHB formation inside the cells of the three isolates by micelle model.
In conclusion, this study is a cornerstone for future use of the tested isolates for PHA production. To the best of our knowledge, this is the first report on large scale production of PHB polymer using A.macrocytogenes isolate P173 and identifying its PHA synthase class III. Moreover, A. baumannii isolate P39 had the highest PHA synthase activity which can be engineered in future studies. Bacillus cereus isolate P83 is a candidate for commercial production of PHB polymer due to cheap carbon source, high production level, and molecular weight of polymer with low polydispersity value.