Identification And Characterization Of Polyethylene-degrading Microorganisms In The Gut Of Galleria Mellonella Larvae

The stable and excellent properties of polyethylene make it widely used in various fields of human production and life.It has become one of the raw materials for plastic products with the largest production and demand in the world in recent years.Its chemical structure is difficult to be degraded and improper disposal methods have caused a large amount of waste polyethylene to accumulate in nature.It continues to pollute water and soil,causing direct or potential harm to plants,animals and even humans.At present,polyethylene has been found in many waters,land,a variety of organisms and foods.The traditional method of processing waste polyethylene will waste resources and cause irreversible pollution to the environment.Therefore,related research on biodegradable polyethylene,such as the search for microorganisms that can degrade polyethylene,has become one of the research hotspots in recent years.Studies have found that the wax moth can feed on polyethylene and decompose it into ethylene glycol by intestinal microorganisms.In the early stage of the laboratory,the larvae of Galleria mellonella were used as the experimental object,and polyethylene was used as the only carbon source.Two strains of LYF-1 and LYF-2 were screened from their intestines,and some physiological and biochemical characteristics were identified.This paper further explored the growth and degradation characteristics of these two bacteria from multiple aspects,and obtained the following results:1.Draw the growth curve of LYF-1 and LYF-2 at 37°C and 28°C,and use the growth curve to determine the stable period of the two bacteria.By measuring the OD₆₀₀ value in the stable phase,the optimum liquid volume,the optimum inoculation volume,and the optimum initial p H range for culturing LYF-1 and LYF-2 under two temperature conditions were explored.The results showed that under the two temperature conditions,LYF-1 and LYF-2 can grow in good condition,but the growth cycle is obviously different.The stable period of the two bacteria is short at 37?but the reproduction speed is faster.At 28?,the stable period of the two bacteria is long but the reproduction speed is slower.The optimum liquid volume for culturing LYF-1and LYF-2 under the two temperature conditions is 30 ml;the optimum p H range is6-7;the optimum inoculum volume does not appear obvious regularity.2.Expand the culture of LYF-1 in the fermentor,and separate the products to obtain the bacteria and extracellular fluid.Bacteria,extracellular fluid and the mixture of the two were respectively smeared on polyethylene film to explore the degradation effects of different components on polyethylene.After 24 hours,it was observed that the materials on the surface of the bacteria group and the mixture of the two groups had dried and cracked,and completely separated from the polyethylene film.The surface solution of the extracellular fluid group evaporated and some substances remained.After 30 days,no changes in the film morphology were found.This experimental method is not feasible.3.The two bacteria LYF-1 and LYF-2 were separately and mixedly inoculated into the M9 liquid medium with linear low-density polyethylene particles as the sole carbon source.And the E.coli inoculation group and the blank group were set at the same time.After 33 days of degradation,the culture medium was made into freeze-dried powder.The group changes of each group of freeze-dried powder and polyethylene particles were analyzed by FTIR and XPS.The results confirmed that the two bacteria have a certain degree of degradability to linear low-density polyethylene.4.The two bacteria LYF-1 and LYF-2 were separately or mixedly inoculated into M9 liquid medium with two common polyethylene films as the sole carbon source.And the sterile M9 liquid group and the dd H₂O group were set as the control group.at the same time.After 12 days of degradation,the changes of each group of polyethylene films were observed by SEM.The results show that LYF-1 and LYF-2can degrade polyethylene film and the effect is obvious.The polyethylene film inoculated with LYF-1 and LYF-2 group has obvious wrinkles and presents an uneven state.Holes or flocculent cracks can be observed below.The polyethylene film of the mixed inoculation of LYF-1 and LYF-2 groups has a stronger fold state,with small holes visible to the naked eye.And a large number of flocculent or flake-shaped fractures around.5.Set up the same grouping experiment as above under the two temperature conditions of 37?and 28?.The growth of the bacteria was evaluated by measuring the OD₆₀₀ value every 2 days and observing the growth of the bacteria on a coated plate,and explored the correlation between it and the degradation effect of polyethylene.The results show that the OD₆₀₀ value cannot fully reveal the growth status of the bacteria.And there is no direct correlation between the OD₆₀₀ value and the growth status of bacteria and the degradation effect of polyethylene.The morphological changes of polyethylene in the inoculated group under the two temperature conditions did not show obvious differences.