Research On Screening Of Two Citrus Dregs Degrading Bacteria And Its Characteristics

China is one of the major citrus producing countries in the world. Citrus is widely cultivated in Jiangxi, Sichuan, Hunan, Hubei, Chongqing and other regions. According to the statistics, the acreage and production of citrus ranked first in the world, which are2.21×106hm2and2.91×107t, respectively. About1.01×106t citrus are processed in China annually; and about5.06x105t citrus dregs are produced during this process. With the development of citrus process industry, the amount of citrus dregs will increase gradually. A large number of untreated citrus dregs after the industrial process are easy to go rotten, heavily polluting the water and soil environment. And also, large amount of odor was produced affecting people’s health seriously. Therefore, the problem of citrus dregs treatment gradually arouses concern, and citrus dregs needs harmless treatment and resource utilization urgently.Recently, main ways to treat citrus dregs include sanitary landfill, citrus dregs drying feed, and active ingredients extraction. Sanitary landfill requires a large amount of arable land, and also produces a large amount of percolate and odor, which pollute environment seriously. Citrus dregs contain large amount of water (>80%) and pectin (20%). Although citrus dregs can be dried for animal feed, the bound water consisting of pectin and water make it difficult of promote due to high energy consumption and high cost. Some effective active substances can be extracted from citrus dregs, such as essential oils, pigments, pectin and falconoid, but the amount of dregs was not effectively reduced with the increment of the volume and type of waste to some extent. These will result in much more difficulties. So present ways of citrus dregs waste treatment not only waste resources, but also seriously pollute the environment, which has become one of the important factors limiting the scale of the development of citrus industry.Citrus dregs contain about20%pectin and15%cellulose, while pectin combined with large quantities of water which increases the difficulty of citrus dregs drying process. Citrus dregs contain cellulose and pectin, increasing the difficulty of microbial degradation. In addition, citrus dregs also contains about0.3%to0.8%of the essential oils, which inhibits the growth of the vast majority of Gram bacteria. Meanwhile, processed citrus dregs contain organic acids, causing pH decrement and then resulting in degradation difficulty for general gicrobial.Therefore, in this study, bacterial isolates L207and L702with high degradation rate were screened out from citrus orchards soil and farmland compost through various separation and screening methods such as high temperature culture, the tolerance of citrus essential oil and Congo red staining. The activity of cellulose and pectin enzyme and the actual ability to degrade citrus dregs were studied, and finally identified its biological status. The results are as follows:1. Isolation of citrus dregs degrading bacteria:the collected soil samples were accessed to different Medias; five bacteria were isolated, which named L106, L202, L207, L309and L702. According to the responding results to different temperature, the tolerance to the citrus essential oil and the cellulose and pectin enzyme activity, L207and L702with high degradation rate were screened out. The results showed that:(1) Determine the inhibition zone diameter of strain using filter paper diffusion method, inhibition zone diameter of L207and L702were found smaller than the other tested strains at the same concentration of essential oils conditions. This indicated that strains L207and L702had better tolerance to essential oils, cultured in a mediums with different concentrations oils using continuous dilution culture method, strains L207and L702were found survived at the maximum essential oil concentration of25.0%and12.5%, it showed that the maximum tolerated concentration of stains L207and L702reached to25.0%and12.5%, respectively. Therefore, L207and L702had better tolerance to the citrus essential oil.(2) Adopting CMC-Na and pectin as sole carbon source, though experimental method of Congo red staining, the hydrolysis zone diameter of strains L207and L702were found significantly larger than the CK. Cultured by shaking flasks for enzyme production, the cellulose enzyme activity of the strains CK, L207and L702, respectively, from big to small were L702, L207, CK. Their cellulose enzyme activities were2.08times and1.31times higher of the CK. And also the pectin enzyme activity of the strains CK, L207and L702from big to small were L207, L702, CK. Their pectinase activities were12.68times and7.30times higher of the CK, respectively. These resul ts showed significant differences (p=0.05). It indicated that strains L207and L702were very excellent strains for degradation of citrus dregs.2. The enzyme-producing conditions of strains L207and L702were optimized. The results showed that the best conditions for producing cellulose and pectin enzyme of stain L207had a high degree of consistency, reached to the maximum under the optimum temperature40℃, the optimal pH value6.0, the best time of degradation enzyme48h to60hand the inoculation was4%(V/V). Under the condition of40℃, the initial pH6.0, for48h to60h,4%(V/V) inoculation, the cellulose enzyme activity of stain L702reached to the maximum. The pectin enzyme activity reached to the maximum under the condition:40℃, the initial pH6.0, for36h to72h,6%inoculation.3. Strain biological identification:by morphological observation, physiological and phylogenetic analysis of16S rDNA, the colonies of the bacteria L207and L702were showed milky and opaque, slightly elevated. They had a moist surface and a little special smell. They were gram-positive with fimbriae. Their spoles were oval, centered and not inflate. Based on the sequence homology comparison, L207and L702were found that similar to E9and DQgbc4in Bacillus licheniformis, respectively. Comprehensive the above characteristics, L207and L702were preliminary identified as the Bacillus licheniformis.4. The actual results of measurement of citrus dregs degradation after vaccinating the two stains L207and L702showed that:after composting50d, the cellulose degradation rate of citrus dregs compost was84.45%, pectin degradation rate was65.93%. Water content reached to16.57%(≤20%) and the total nutrient (N+P2O5+K2O) reached to4.71%(≥4.0%), completely reached the national standard of organic fertilizer (NY525-2002). This showed that the strains L207and L702had strong degradation capacity of citrus dregs.