Research And Development Of Microbial Agents For Reusing Solid-liquid Wastes And Study On Relationship Between N-fixing Bacteria And Soil Properties

For the benefit of improving nutrition and resistance to fungi pathogenesis for plant, accelerating maturity of compost, microbial agents are used widely in agriculture and in thermophilic composting of livestock or poultry wastes. It plays important roles in protecting agricultural ecological environment and enhancing agricultural production. But quite high production cost and unstable inoculation effect of microbial agents are the main obstacles for its application. On the other hand, difficulties of pollution disposal increase for large amounts of high concentration organic wastewater produced in food industry. Monosodium glutamate wastewater (MGW) characterized with low pH, high COD and BOD value, high SO₄^2-, high cell concentration and large amount of discharged volume is difficult to be disposed completely for manufacture. Many components in MGW, e.g. N, P, K and trace elements, are high quality nutrition resources for plants and microorganisms. The difficulty of pollution disposal might be alleviated if MGW is used to produce microbial agents and /or develop nutrient solutions for medium culture at a certain extent, which reuse MGW as nutrient resource of plant and reduce the cost of producing microbial agents. In this article, cheap culture media of isolated functional microorganisms for composting and associated nitrogen fixing bacteria was developed using high concentration organic wastewater of food industry as nutrient resources. The effect of composting microbial agents was verified in three composting experiments. The feasibilities of using improved MGW as nutrient solution for tomato were also studied. To understand the unstable inoculation effect of microbial agents in field, the relationship between the inoculation effect of exotic nitrogen fixing bacteria and soil properties was discussed. Main results are listed as follow:1. 10 candidate strains capable of fibre-degrading were screened using selective media of bacteria, mildew, fungi, actinomyces and microzyme at 30℃or 45℃from compost, chicken manure and vegetable soils; and another 9 candidate strains capable of deodorization were screened using culture media of mildew, actinomyces and microzyme and carboxymethyl cellulose sodium at temperature of 30℃,45℃or 75℃from the same source. According to the further studies on the functions of fibre-degrading, deodorization and plant pathogenic fungi resistance, 8 strains of B.Licheniformis, B.Subtilis, B.macerans, et al. capable of fibre-degrading, deodorization or plant fungi disease resistance were screened for the exploitation of thermorphilic composting fermentogen from 15 strains isolated or collected by our own lab. The major function of each stain was listed in table 2-13.2. The recipes of basic culture media for these 8 stains were selected by L₉(3⁴) orthogonal design experiments for 3 factors, e.g. MGW, bean products wastewater (BPW) and cane sugar. Based on L₁₆(2¹⁵) orthogonal design experiments of 12 nutrient elements, the former basic culture media recipes were optimized by additive macro-elements and/or microelements. It help us find the way to produce low cost microbial agents by using MGW and BPW. The application of national invention patent for these recipes has been accepted (Application Number: 200510049704.9).3. Three thermophilic composting experiments for chicken manure were conducted for testing the effects of low cost microbial agents on composting process. Results of autumn composting experiment show the temperature of compost heap inoculated with 5 single strain agents and 2 multiple strains agents is significantly higher than that of uninoculated control treatment. Bacillus lieheniformis, Bacillus macerans and No.2 combined agent are especially benefit for enhancing the temperature of compost heap. Effects of 9 single strain agents and their combined agent on enhancing compost heap temperature were compared with 2 commercial microbial agents in winter compost experiment. Six strains including Bacillus lieheniformis, Bacillus subtilis, Bacillus macerans, Trichoderma reesei, CMC-W70-S and CS-R30-P showing better and/or the same heap temperature enhancing effects compared with commercial products were screened for next composting experiment. A composting experiment was further conducted to confirm the effect of the combined agent with former selected 6 strains. The result indicated that the combined agent showed the same or better effects of enhancing compost heap temperature and protecting ammonia from volatilization as commercial product. Furthermore, content of total N, P, K and organic matter of organic fertilizer are higher than that of the commercial agent. It predicts that this agent with selected functional microbial strains prepared using high concentration organic wastewater of food production as nutrient resources is capable of being applied in thermophilic composting of animal manure.4. The feasibility of MGW as main nutrition resource in nutrient solution of tomato medium culture and the effect of microbiological agents such as associative nitrogen-fixing bacteria and Bacillus licheniformis inoculants prepared by organic wastewater, on growth of watermelon and tomato were studied by tomato potted planting experiment, greenhouse drop-irrigation experiment and watermelon field experiment.â‘ The results of tomato potted planting experiment indicated that plant height, leaf number and stem diameter were significantly increased in MGW treatment compared with the traditional nutrient solution (TNS) treatment. Furthermore, the first flowering stage was earlier and the yield of tomato was higher than those of the latter treatment. Meanwhile, there are no siginificant difference found in the eclectrical conductivity (EC) of the media between the MGW treatment and the TNS treatment.â‘¡The results of the second tomato medium culture experiment applying MGW combined with W12 inoculant indicated that higher nitrogen contents of tomato roots and leaves were abtained in the W12 inoculating treatment compared with non-inoculated treatment. The nitrogen contents of tomato roots and leaves in MGW treatment were higher than those in TNS treatment. Furthermore, the positive interaction was showed between application of MGW and inoculated W12 on the nitrogen contents in tomato shoots. In conclusion, MGW not only provide the nutrients for plant, but also for W12. The tomato yield of the MGW treatment is higher than that of TNS treatment. Senescence of the older leaves was delayed in MGW treatment compared with TNS treatment. Although the culture medium pH is a little lower and the electric conductivity is a bit higher in MGW treatment than those in TNS treatment after the experiment finished, but both are still suitable for tomato growth.â‘¢Results of meidium culturing cherry tomato experiment in greenhouse showed that MSW as drip irrigation nutrition can improve the nutrition supply of tomato and significantly enhancing the content of Chlorophyll and carotenoid of leaves. No significant difference is found in the antiaging factors, e.g. SOD, MDA and POD between traditional inorganic nutrition solution and MSW drip irrigation treatments. Content of soluble proteins of tomato leaves in MSW treatment is significantly higher than that in the inorganic nutrition treatment. The total amino acid content, total sugar content and ratio of sugar to acid of tomato fruits in MSW treatment were significantly enhanced while total acidity is reduced compared with the inorganic nutrition treatment. Therefore, application of MSW can improve the edibility and nutrient quality of fruits besides the same yield obtained as the inorganic nutrition treatment. In addition, yield increased after inoculating Bacillus lieheniformis agent prepared with MSW in the watermelon field experiment. In conclusion, MSW used as the main nutrition resource in nutrient solution for tomato medium culture is suitable for plant growth and can obtain reasonable yield and higher product quality, and may be capable of enhancing microbial agent inoculation effects. A national invention patent for this technique was obtained (patent Number: ZL 200510049612.0).5. In order to understand the instability of microbial agent inoculating effect in agriculture production, the relationship between soil chemical and biological traits and competitive ability of exotic nitrogen fixation bacteria against soil indigenous bacteria was studied. A competitive growth experiment between the Rifampin resistant associative nitrogen fixation bacteria W12-R and soil indigenous bacteria in the extracts of three soils with distinguishing properties was conducted. Results showed that there was no significant difference found in the counts of W12-R when pure cultured in three soil extracts, however growth of W12-R were inhibited in a wide extent when cultured together with soil indigenous bacteria, especially in the extract of Honghe soil having lowest contents of available nutrient. However, supplement with some carbon source could alleviate the inhibition in a various degree, particularly in the Honghe soil extract where the growth increment of W 12-R reverted to the level of pure culture. Nitrogenase activities of W12 pure cultured or cultured together with indigenous bacteria of three soils were determined. It was found that nitrogenase activities of co-culture systems sharply declined contrast to that of pure culture system and nitrogenase activities within three co-culture systems were significantly different. The lowest nitrogenase activity was detected in the co-culture system of Xindai soil which had the highest levels of total nitrogen content, organic matter and microbial biomass and the highest was found in Honghe soil co-culture system. It indicated that soil indigenous bacteria could not only compete for nutrients with exotic inoculated nitrogen fixing bacteria but also affect on their nitrogenase activity. It can be found that the higher the contents of soil NH₄⁺-N, microbial biomass C and microbial biomass N as well as amounts of soil indigenous bacterial, fungi and actinomycete, the lower the nitrogenase activities in co-culture systems based on analysising the relationship between these properties. In conclusion, positive inoculation effect of exotic nitrogen fixation bacteria angent might be obtained when inoculated in soils with lower content of NH₄⁺-N, lower fertility and microbe biomass, especially supplemented with proper carbon source.6. The nitrogenase activities of indigenous nitrogen fixing bacteria of 27 soils (NAS) and of exotic nitrogen fixing bacteria W12 together with indigenous microbes of these soils (NA (S+W12)) were tested. The inoculating effects of W12 was estimated by the difference between NA(S+W12) and NAS, denoted as NAD. Multiple statistical analysis results showed that among 13 soil properties tested, only the content of soil available Cd is significant partial negative correlated with NAS. In the co-culture system of exotic nitrogen fixing bacteria W12 and indigenous microbes, significant negative partial correlation between NA(S+W12) or NAD and soil NH₄⁺-N content, and significant partial positive correlation between NA(S+W12) or NAD and soil available Cr were found. Although, NA(S+W12) is significantly positive correlated with NAS, significant negative correlation was showed between NAD and NAS. It indicates that part of the fixed nitrogen in co-culture system is contributed by indigenous microbes and higher NAS may be unfavorable for inoculating effect of exotic nitrogen fixing bacteria. 27 soils can be classified to 3 categories according to their inoculation effect by hierachical cluster analysis. Positive inoculation effect of W12 found in the first group may be caused by the highest content of soil available Cr (positive correlated factor of inoculating effect) and moderate content of soil NH₄⁺-N content and NAS (negative correlated factors). The negative inoculation effect of W12 showed in the second group may be due to its highest content of soil available Cd. The higher NAS might be the cause of the negative inoculation effect in the third group. According to the curve between NAD and soil available Cr content or soil NH₄⁺-N content, the stress threshold of soil available Cr and soil NH₄⁺-N on W12 are 2μg kg^-1 and 20mg kg^-1, respectively. That is to say, the positive inoculation effect can hardly be obtained when the content of soil available Cr is lower than 2μg kg^-1 or soil NH₄⁺-N content higher than 20mg kg^-1.