Production of Biodegradable Polymer from Agro-Wastes in Alcaligenes sp. and Pseudomonas sp.

The present study was aimed to evaluate the suitability of agro-wastes and crude vegetable oils for the cost-effective production of poly-ß-hydroxybutyrate (PHB), to evaluate growth kinetics and PHB production in Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 with these carbon substrates and to study the biodegradation of PHB accumulated by these cultures. Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1 accumulates higher amounts of PHB corn (79.90% of dry cell mass) and rice straw (66.22% of dry cell mass) medium respectively. The kinetic model suggests that the Pseudomonas sp. RZS1 follows the Monod model more closely than A. faecalis RZS4. Both the cultures degrade their PHB extract under the influence of PHB depolymerase. Corn waste and rice straw appear as the best and cost-effective substrates for the sustainable production of PHB from Alcaligenes faecalis RZS4 and Pseudomonas sp. RZS1. The biopolymer accumulated by these organisms is biodegradable in nature. The agro-wastes and crude vegetable oils are good and low-cost sources of nutrients for the growth and production of PHB and other metabolites. Their use would lower the production cost of PHB and the low-cost production will reduce the sailing price of PHB-based products. This would promote the large-scale commercialization and popularization of PHB as an ecofriendly bioplastic/biopolymer.

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7.

There are numerous reports on poly-ß-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.

Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard.

Poly-ß-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymerases from isolates obtained from plastic-contaminated sites that reflect the potential of the source organism is scarce. In this study, we evaluated the production of extracellular PHB depolymerase from Microbacterium paraoxydans RZS6 isolated from the plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters (GC-FAME), and BIOLOG method. Ithydrolyzed PHB on minimal salt medium (MSM) containing PHB as the only source of carbon. The isolate produced PHB depolymerase at 45°C during 48 h of incubation. The enzyme was purified most efficiently using octyl-sepharose CL-4B column, with the highest purification yield of 6.675 Umg-1mL-1. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ ions but inhibited by Fe2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg2+ ions, that showed optimum enzyme activity at 30°C (mesophilic) and under neutrophilic (pH 7) conditions. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield by 0.809 UmL-1. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled the PHB depolymerase of Aureobacterium saperdae. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase having potential of degrading PHB under diverse conditions.

Statistical-based optimization and scale-up of siderophore production process on laboratory bioreactor.

We report the enhanced production of siderophore in succinate medium by applying two-stage statistical approach, i.e., Plackett-Burman design and response surface methodology (RSM) using central composite design (CCD). In the first stage of optimization, out of 11 variable components of succinate medium, succinic acid, pH and temperature were found as significant components that influenced the siderophore production in Pseudomonas aeruginosa RZS9. The second stage of RSM using CCD consisted of optimizing the concentrations of the variables. Here, 0.49 g/100 ml concentration of succinic acid, pH 7.08 and temperature of 27.80 °C yielded the maximum (68.41 %) siderophore units. All the significant components exhibited quadratic effect on siderophore production. The F value of 28.63, multiple correlation coefficient (R 2) of 0.9626, percent coefficient of variation of 8.81 values indicated that the model was significant and that the experimental data was satisfactorily adjusted to the quadratic model. During validation of these experiments, 6.10 % increase in siderophore yield was obtained. Scale-up of this protocol optimized at shake  flask level up to 5 L-capacity reactor further enhanced the siderophore yield. We claim it to be the first report on statistical optimization of siderophore production by P. aeruginosa RZS9.

Stenotrophomonas sp. RZS 7, a novel PHB degrader isolated from plastic contaminated soil in Shahada, Maharashtra, Western India.

This paper reports an isolation and identification of novel poly-ß-hydroxybutyrate (PHB) degrading bacterium Stenotrophomonas sp. RZS 7 and studies on its extracellular PHB degrading depolymerase enzyme. The bacterium isolated from soil samples of plastic contaminated sites of municipal area in Shahada, Maharashtra, Western India. It was identified as Stenotrophomonas sp. RZS 7 based on polyphasic approach. The bacterium grew well in minimal salt medium (MSM) and produced a zone (4.2 mm) of PHB hydrolysis on MSM containing PHB as the only source of nutrient. An optimum yield of enzyme was obtained on the fifth day of incubation at 37 °C and at pH 6.0. Further increase in enzyme production was recorded with Ca2+ ions, while other metal ions like Fe2+ (1 mM) and chemical viz. mercaptoethanol severally affected the production of enzyme.