Preparation Of Tobacco Residue-based Microbial Fertilizer By Solid-state Cultivation Of Mixed Bacteria

About 25% tobacco was abandoned in the processing and a large number of unqualifiedfresh leaves was discarded,which is a great waste of resources without utilization.In this thesis,the residue of tobacco was used as medium of solid-state cultivation to produce microbial fertilizer,in which the active ingredients have been extracted.The culture conditions of mixed strain of Bacterial A and Bacterial A-Bacterial B-Fungal A were studied,and the effects of residual ethanol and raffinate addition were explored.Finally,the preparation process at laboratory scale was determined.With tobacco residue as solid-state cultivation medium,the strains of Bacterial A were screened.The effects of the mixing method,raw material,layer thickness and the water content on the solid culture of Bacterial A were investigated.The conditions for the solid-state culture of Bacterial A were as follows: 500 m L conical flask containing 60 g tobacco residue with 58% saturated water content,10% inoculation,culture temperature 37?C,culture time 11~12 days.The spores in final product was over 1012 cfu/g.With tobacco residue as solid-state cultivation medium,Bacterial B and Fungal A were screened.The effects of inoculation ratio of three microorganisms(Bacterial A,Bacterial B and Fungal A),culture temperature,layer thickness on the solid-state culture of the mixed bacteria were investigated.The conditions for the solid-state culture of mixed bacteria fertilizer as follows: The seeds of Bacterial A / Bacterial B / Fungal A were cultured separately,and mixed at a ratio of 6:3:1.In a 250 m L conical flask containing 35 g tobacco residue(saturated water content 58%)with the layer thickness of 3.5 cm,the mixed seed was inoculated at an amount of 10% of the solid medium,then culutrued at room temperature(15?C ~25?C)for 10 days.The spores of each bacterium in final prodcut reached 1011 cfu/g.The number of spores of each bacterium could reach 109 cfu/g when the 500 m L conical flask containing 70 g tobacco residue with the layer thickness of 4.5 cm was applied.In the pretreatment of tobacco for active ingredient extraction,ethanol was used and there was extraction raffinate produced during extraction processes,therefore the effects of residual ethanol and raffinate addition on bacterial growth were studied.The results showed the residue ethanol(1%~3%)inhibited the growth of bacteria.However,the total number of spores still reached 106 cfu/g after 8-day cultivation at 8?C~15?C.When the culture mediumwas prepared withthe 50%~60% raffinate solution,the spore number of Bacterial A reached108 cfu/g after 7-day cultivation at 8?C~15?C.With fresh tobacco residue as solid-state cultivation medium of mixed bacteria,in which the active ingredients had have been extracted,it was found that Bacterial A growed much faster.The spore number of Bacterial A was over 109 cfu/g after 7-day cultivation at8?C~15?C.Under the conditions without moisture control,10 g dry residue can produce about 20 g of bacterial fertilizer with a water content about 50%.When 1 tons of tobacco were extracted by nicotine and perfume,500 kg dry residue can be obtained,thus 1 tons wet bacterial fertilizer(water content 50%)can be produced.