Study On Production Of Polyhydroxyalkanoates By Halophiles Using Waste Silkworm Excrement

Polyhydroxyalkanoates(PHAs)are a class of biodegradable plastics with similar material properties as traditional petroleum-based plastics(e.g.polypropylene),and are ideal alternatives to traditional plastics.The synthesis of PHAs by microbial fermentation is the mainstream of industrial-scale production of PHAs,but the high cost of fermentation production restricts the wide application of PHAs.Therefore,this study selected cheap,widely available,and renewable raw materials as the carbon source for PHAs synthesis,and adopted open fermentation without sterilization by taking advantage of halophiles,which effectively reduced the cost of PHAs production and played an important role in realizing the recycling of waste and reducing the environmental pollution.The silkworm excrement is a typical agricultural waste that is hardly biologically reused.However,partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate PHAs.Therefore,this study reported for the first time the model of PHAs accumulation by halophiles using waste silkworm excrement as the carbon source for open fermentation.First,in this study,a total of 14 different microorganisms were isolated from the waste silkworm excrement,of which10 were bacteria and four were archaea.By using the Nile red staining and gas chromatography assays,two archaea and two bacteria were shown to accumulate PHAs,and further studies revealed that the two haloarchaea were better able to accumulate PHAs using silkworm excrement as the sole carbon source.After open fermentation,the two archaea Haloarcula hispanica and Natrinema altunense accumulated up to 0.31 ± 0.01 g/L and 0.12± 0.02 g/L of PHB,respectively,in the medium with silkworm excrement as the sole carbon source,and separately up to 0.81 ± 0.05 g/L and 0.58 ± 0.04 g/L of PHBV after 0.5%glucose addition.In addition,the CFU experiments revealed that there were more than 60%target PHAs producing haloarchaea cells at the time of the highest PHAs production,and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells(to nearly 100%).Our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement,effectively.The previous study found that haloarchaea had a better utilization effect on the silkworm excrement.Therefore,this study investigated the PHAs accumulation of the haloarchaea Haloferax mediterranei ATCC 33500 using the silkworm excrement in open fermentation.It was found that the strain ATCC 33500 could accumulate PHBV using the silkworm excrement as the sole carbon source.While silkworm excrement and glucose were mixed with a 1:1 carbon content ratio,the ability of the strain ATCC 33500 to accumulate PHBV was significantly improved compared with glucose as the sole carbon source,with a maximum yield of 1.73 ± 0.12 g/L.When the Na Cl concentration was at approximately 15%,an open fermentation without sterilization was set up using silkworm excrement as the carbon source.It was noteworthy that the addition of the silkworm excrement could increase the 3HV content of the fermentation product PHBV regardless of the sterilization or non-sterilization fermentation conditions,and the 3HV content was as high as 16.37 ± 0.54 mol % with the silkworm excrement as the sole carbon source.Further study on the effect of the silkworm excrement on the 3HV synthesis-related genes of PHBV by the strain ATCC 33500 revealed that the expression of the key gene mgl/met C in pathway II was significantly up-regulated relative to the control group after short-term or long-term stimulation with the silkworm excrement.Therefore,the experimental results showed that the mechanism of the enhancement of 3HV content was that silkworm excrement mainly promoted the aspartate/2-oxobutyrate pathway(pathway II),which increased the content of 2-ketobutyric acid,and then further increased the content of propionyl-Co A through pyruvate or α-ketoglutarate: iron oxidoreductase decarboxylation to produce propionyl-Co A,thus improved the 3HV content.In summary,this study not only provides a good model strain and fermentation mode for the production of PHAs by open fermentation of agricultural wastes,which solves the problem of the high cost of PHAs production,but also,based on the strain Haloferax mediterranei ATCC 33500,explains the mechanism of increased the 3HV content in PHBV synthesized by the strain ATCC 33500 using the silkworm excrement at the gene transcription level for the first time,which provides novel strategies for adjusting the content of 3HV in PHBV and improving its material properties.