Preparation of Penicillin Mycelium Fertilizer and Its Environmental Safety Study Following Soil Application

Author:Wang Bing

Supervisor:liu hui ling


Degree Year:2018





Penicillin mycelium dreg(PMD)is a kind of waste residue dreg produced in penicillin fermentation process.Although PMD is rich in bacterial protein and other nutrients,it owns potential risk of drug-resistant bacteria during the re-utilization due to its drug residue.Given that there is no safe disposal method and risk assessment for PMD,we try a hydrothermal pretreatment method to remove the residual penicillin in PMD.Then after the pretreatment,the penicillin mycelium fertilizer(PMF)were studied deeply on its soil environment behavior in order to lay the ground for the recycle and utilization of PMD.In order to optimize the parameters of thermal method to remove the residual penicillin,this study investigated the effect of temperature and time on the stability of penicillin in standard solution and penicillin mycelium dreg.In order to degrade penicillin G in PMD,the optimum temperature and time was 100 ℃,2 h.The removal rate of penicillin G is about 98%.Then LC/MS/MS was used to qualitatively analyse the hydrothermal degradation process of penicillin.Under the negative ion mode scanning,there were four kinds of special material peaks,which were 307,333,351 and 333,respectively.The 4 kinds of material peaks were inferred as decarboxylation penicillium thiazole acid,penicillin G,penicillium thiazole acid and formic acid products of penicillin.To evaluate the soil environmental behavior of PMF,a lab scale of experiment was carried out to study the soil physical and chemical properties,microbial properties and antibiotic resistance genes.It was found that in PMD and PMF treatment,the initial content of penicillin G was respectively 10 ~ 32 mg/kg and 26 ~ 132 mg/kg,and then rapidly reduced,disappeared in the sixth and eighth days.Three-dimensional fluorescence spectrometry(EEM)shown that the characteristic peak of protein matters and microorganism metabolites appeared in the initial stage,and then gradually decreased.The corresponding excitation /emission wavelength was 225/343 nm and 280/347 nm.The two peaks associated with humic acid appeared in the later,and gradually increased.The corresponding excitation/emission wavelength was >250 nm and >380 nm,respectively.The seed germination index(GI)revealed that the application of PMF significantly reduced GI values at early stage,which decreased with the increase of fertilizer rate.late GI value increased gradually,and finally exceeded the GI of blank soil.Dilution plate method was used to determine the numbers of bacteria,fungi,actinomycetes,which can reflect the effect of PMF on microbial activity.It was found that PMF promoted the growth of microorganism,but PMD inhibited the growth of bacteria at the early stage.With the high-throughput sequencing of 16 s r RNA gene V4 area of bacteria,the effect of PMD and PMF on soil microbial community structure was determined that both of them significantly changed the original indigenous microbial species.The ratio of deformation bacteria decreased,and the ratio of actinomycetes increased.The bacterial diversity significantly reduced.The OTUs dilution curve showed that bacterial diversity increased first and then decreased.To study the problem about whether the application of PMD introduces or causes antibiotic resistance genes(ARGs),the high through real-time fluorescent quantitative PCR was used to study the diversity of soil microbial ARGs.It was found that 6 of beta lactam type of resistance genes(ARGs)and 1 of transposase gene(MGEs)was detected in the original PMD and PMF samples.In soil samples,PMD significant changed the structure of soil microbial ARGs,and increased the abundance and diversity of ARGs.On the contrary,PMF did not significantly change the abundance and diversity of ARGs,which was similar with the ARGs of blank soil.Twelve ARGs was detected in blank soil,9 ~ 14 ARGs in treated-PMD and 15 ARGs in PMD.Fold change(FC)was studied and found that the treated-PMD did not significantly influence the soil ARGs,while more reducing ARGs(FC < 1)were detected in PMF treatment.Finally,to evaluate the practical application of penicillin mycelium fertilizer(PMF)as organic fertilizer,a greenhouse experiment was carried out with crops planting.Its effects on soil properties,phytotoxicity,microbial community composition,enzyme activities and growth of snap bean in greenhouse were investigated.As the results shown,p H,total nitrogen,total phosphorus,total potasisum and organic matter of soil with PMF treatments were generally higher than CON treatment.In addition,fertilized PMF had not caused heavy metal and residual drug pollution for modified soil.But the lowest GI values were recorded at PMF8(GI<0.3)at early fertilizing days and it indicated that severe phytotoxicity appeared in the PMF8 modified soil.Results of microbial population and enzyme activities illustrated that PMF was rapidly decomposed and the decomposition process significantly affected microbial growth and enzyme activities.The phytotoxicity of PMF modified soil disappeared at the late fertilization time.The appropriate amount of PMF reduced the crop of SOD,POD,CAT,APX activity and MDA content,indicating that it did not induce adversity stress on crops.But excessive amount of PMF induced adversity stress on crops because various antioxidant enzyme activity were very high.PMF1 was regarded as the optimum amount of PMF dose with the scores order of principal component analysis.Plant height and plant yield of snap bean were remarkably enhanced with optimum PMF dose.