固液废弃物资源化功能微生物制剂研发及外源固氮菌与土壤特性关系研究
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摘要
微生物制剂广泛应用于禽畜粪高温堆肥及农业生产，以促进堆肥腐熟、增加作物养分供应或增强作物抗病性，对保护农业生态环境和农业增产增收具有重要作用。然而，由于生产和使用成本过高以及接种效应的不稳定限制了农用微生物制剂的推广应用。另一方面，大量高浓度食品工业有机废液，如谷氨酸发酵液经提取谷氨酸后剩下的味精废液，具有低pH、高COD和BOD、高SO_4~(2-)、高菌体含量和排放量大的特点，导致治污达标困难，带来巨大环境污染治理压力。其含有的大量N、P、K营养和多种微量元素能为微生物和植物生长提供丰富优质的营养源。因此，将味精废液等食品工业有机废水作为微生物制剂和基质栽培营养液的营养源开发利用，可以减轻其环境污染治理压力，实现其营养资源的再循环利用，并降低微生物制剂和基质栽培的生产成本。为此，本文在分离、筛选获得高效功能微生物菌株的基础上，以高浓度食品工业有机废水为营养源，研究了畜禽粪廉价发酵菌剂和联合固氮菌接种剂等农用微生物制剂的培养基配方和应用效果；研究探讨了改性味精废液作为番茄基质培营养液的可行性；针对微生物制剂接种效果的不稳定性问题，着重探讨了外源固氮菌接种效果与土壤因子的相互关系。取得主要结果如下：
(1)从市售发酵有机肥、鸡粪及菜园土壤等分离源，利用细菌、霉菌、真菌、放线菌和酵母菌选择性培养基，分别在30℃、45℃培养温度下，共分离获得了10个纤维素降解功能候选分离物；利用霉菌、放线菌、酵母菌和羧甲基纤维素钠培养基，分别在30℃、45℃和70℃培养温度下，共分离获得9个除臭功能候选分离物。进一步通过纤维素降解能力、除臭能力和植物病原真菌抗性测定，从15株自行分离或实验室收藏的菌株中筛选出地衣芽孢杆菌、枯草芽孢杆菌、浸麻芽孢杆菌、新木和里氏木霉等8株具有或兼具降解纤维素、除臭和抗病等多重功能的微生物作为高温堆肥发酵菌剂开发的候选菌株。
(2)通过味精废液、黄泔水和蔗糖3因子3水平L_9(3~4)正交设计试验，筛选出8个供试畜禽粪发酵候选菌株的3种营养源基础培养基配方；并通过12种营养元素的L_(16)(2~(15))正交设计试验，在上述3种营养源基础培养基配方的基础上筛选出各候选菌株添加常量和微量元素的优化配方。结果表明，利用味精废液、黄泔水等食品工业有机废水作为功能菌的基本营养来源，并适量添加常量和微量元素来生产廉价微生物菌剂是可行的。培养基配方申请国家发明专利(申请号：200510049704．9)，已通过初审进入实审。
(3)通过鸡粪高温堆肥发酵试验，研究了自行制备的廉价发酵菌剂的高温堆肥效果。秋季堆肥试验结果表明，5个单一功能菌剂及它们的2个复合菌制剂接种处理，相对于不接种对照，加速肥堆升温效果显著，其中地衣芽孢杆菌、浸麻芽孢杆菌及混合菌剂2的升温作用更为明显。冬季堆肥试验，以2个市售发酵菌剂和不接种空白处理为对照，比较了接种9个单一菌剂及它们复合菌剂的堆肥升温效果，筛选出肥堆升温和促腐效果达到或超过酵素菌的菌株6个：地衣芽孢杆菌、枯草芽孢杆菌、浸麻芽孢杆菌、里氏木霉、CMC-W70-S、CS-R30-P。春季堆肥试验结果表明，采用6个菌株制备的混合菌剂接种处理的肥堆升温、保氮、pH和有机肥全氮磷钾含量及有机质含量达到或超过市售发酵菌剂的接种效果，表明本研究开发的利用有机废液生产的发酵菌剂在禽畜粪高温堆肥中具有实际应用价值。
(4)通过番茄基质培盆栽试验、大棚滴灌试验和西瓜田间试验，研究了改性味精废液作为番茄基质栽培营养液的可行性以及采用有机废液制备的联合固氮菌W12和地衣芽孢杆菌等微生物制剂对西瓜、番茄的接种效应。①番茄基质培盆栽试验结果表明，浇施味精废液处理可显著增加番茄株高、叶片数和茎粗，促进其营养生长，并使番茄提早开花，有利于生殖生长，尽管始花期恰逢连阴雨天气影响初期花的挂果，但产量仍略高于完全营养液处理。而且，栽培基质的电导率与浇施完全营养液处理无显著差异。②味精废液与W12菌剂联用促生效果基质培盆栽试验结果表明，番茄茎、叶的全氮量表现为接种W12处理极显著高于不接菌对照，浇味精废液处理极显著高于其它两个营养液，且番茄茎全氮含量在基质接种方式与不同营养液间的交互作用极显著，说明浇施味精废液不仅可直接为番茄植株提供营养，还可以为W12提供丰富的营养源。浇施味精废液的番茄产量显著高于完全营养液处理，而且生长后期的下位叶黄化程度明显比后者减轻。虽然浇施味精废液的栽培基质pH略低于完全营养液处理，基质的电导率略高于后者，但均在番茄生长安全pH和电导率范围内。③樱桃番茄基质培大棚滴灌味精废液试验结果，进一步证实了味精废液能改善番茄叶片的氮素营养，显著提高其叶绿素和类胡萝卜素含量。滴灌味精废液的番茄功能叶抗衰老相关因子SOD、MDA、POD和常规营养液无显著差异，却显著提高叶片可溶性蛋白含量，说明有利于提高番茄叶片的抗逆性。滴灌味精废液不仅取得与滴灌常规无机营养液相同的产量，而且果实总氨基酸增加30％，总糖度及糖酸比显著提高，总酸度明显降低，明显改善番茄果实的鲜食风味和营养品质。此外，微生物菌剂对西瓜接种田间试验结果表明，接种采用味精废液制备的地衣芽孢杆菌菌剂能使西瓜产量有所提高。综上所述，经过改性的味精废液可以作为基质培番茄营养液，且能与联合使用的功能微生物菌剂产生显著的正交互作用，提高功能微生物接种效果。该项技术已获国家发明专利授权(ZL200510049612．0)。
(5)为探讨土壤化学和生物学性状与外源固氮菌对土著菌竞争力的关系，采用联合固氮菌W12的利福平诱导抗性菌株W12-R，设计了W12-R在3个熟化度和肥力水平差异悬殊的土壤浸提液中与土著菌的竞争生长试验。结果表明，W12-R在3个土壤浸提液中单独培养时生长量无显著差异，但与土著菌共培养时，W12-R的生长受到了不同程度的抑制，其中以在有效养分含量最低的洪合土壤浸提液中受到的竞争抑制最大。补充适量碳源可不同程度削弱这种抑制，其中在洪合土壤浸提液中最为突出，使W12-R生长量回复到纯培养水平。比较在无氮培养基中W12单独以及分别与各供试土壤土著菌共培养系统中测得的固氮酶活性，发现共培养系统的固氮酶活性相对于纯培养系统大幅下降，而且三个土壤的共培养体系的固氮酶活性差异极显著(p=0.0001)，其中以总氮、有机质、微生物量最高的新埭土壤共培养系统最低，而各项土壤特性指标相对较低的洪合土壤共培养系统最高。可见，外源固氮菌W12与土著菌共存时，土著微生物不仅对其生长造成营养竞争抑制，对其固氮酶活性也产生了显著的影响。进一步分析土壤NH_4~+-N含量、微生物量碳、微生物量氮、TOC以及土著细菌、真菌和放线菌数量与共培养系统固氮酶活性的关系，结果表明土壤NH_4~+-N含量和土著微生物数量越高，W12与土著菌共培养系统的固氮酶活性降低越多。由上述结果可见，对微生物量较少，肥力较低，特别是NH_4~+-N含量较低的土壤接种外源固氮菌W12，并利用固、液有机废弃物补充适当的营养源，将有利于提高其接种效果。
(6)多元统计分析结果表明，土壤土著菌固氮酶活性NAS仅与13项土壤性状指标中的土壤有效态Cd呈显著偏负相关；外源菌W12与土著菌共培养系统的固氮酶活性NA(S+W12)及W12接种效应估计值NAD(NA(S+W12)-NAS)分别与土壤NH_4~+-N、有效态Cr含量呈显著的负偏相关和正偏相关；NA(S+W12)与NAS呈显著的正相关，而NAD却与NAS呈显著的负相关，表明接种W12后共培养系统的总固氮量有一部分是来自土著固氮菌的贡献，但较高NAS却不利于提高外源固氮菌W12的接种效应。通过系统聚类分析将27个供试土样分为外源固氮菌W12接种效应明显不同的3个类型，其中，第一类由于接种效应正相关因子土壤有效Cr水平较高，而负相关因子NH_4~+-N、NAS居中等水平，因而表现为正种效应；第二类因土壤有效态Cd过高，表现为负接种效应；第三类可能由于NAS过高而具有负接种效应。根据外源固氮菌W12接种效应估计值NAD与其正相关因子土壤有效态Cr含量及负相关因子土壤NH_4~+-N含量的关系曲线，初步发现在供试土壤范围内，两者对外源固氮菌W12接种效应产生胁迫的临界值分别为2μg kg~(-1)和20mg kg~(-1)，即土壤有效态Cr含量低于2μg kg~(-1)和／或土壤NH_4~+-N含量高于20mg kg~(-1)将抑制外源固氮菌W12的接种效应发挥。
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 SO_4~(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 L_9(3~4) orthogonal design experiments for 3 factors, e.g. MGW, bean products wastewater (BPW) and cane sugar. Based on L_(16)(2~(15)) 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.①The 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.②The 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.③Results 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 NH_4~+-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 NH_4~+-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 NH_4~+-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 NH_4~+-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 NH_4~+-N content, the stress threshold of soil available Cr and soil NH_4~+-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 NH_4~+-N content higher than 20mg kg~(-1).
(1)从市售发酵有机肥、鸡粪及菜园土壤等分离源，利用细菌、霉菌、真菌、放线菌和酵母菌选择性培养基，分别在30℃、45℃培养温度下，共分离获得了10个纤维素降解功能候选分离物；利用霉菌、放线菌、酵母菌和羧甲基纤维素钠培养基，分别在30℃、45℃和70℃培养温度下，共分离获得9个除臭功能候选分离物。进一步通过纤维素降解能力、除臭能力和植物病原真菌抗性测定，从15株自行分离或实验室收藏的菌株中筛选出地衣芽孢杆菌、枯草芽孢杆菌、浸麻芽孢杆菌、新木和里氏木霉等8株具有或兼具降解纤维素、除臭和抗病等多重功能的微生物作为高温堆肥发酵菌剂开发的候选菌株。
(2)通过味精废液、黄泔水和蔗糖3因子3水平L_9(3~4)正交设计试验，筛选出8个供试畜禽粪发酵候选菌株的3种营养源基础培养基配方；并通过12种营养元素的L_(16)(2~(15))正交设计试验，在上述3种营养源基础培养基配方的基础上筛选出各候选菌株添加常量和微量元素的优化配方。结果表明，利用味精废液、黄泔水等食品工业有机废水作为功能菌的基本营养来源，并适量添加常量和微量元素来生产廉价微生物菌剂是可行的。培养基配方申请国家发明专利(申请号：200510049704．9)，已通过初审进入实审。
(3)通过鸡粪高温堆肥发酵试验，研究了自行制备的廉价发酵菌剂的高温堆肥效果。秋季堆肥试验结果表明，5个单一功能菌剂及它们的2个复合菌制剂接种处理，相对于不接种对照，加速肥堆升温效果显著，其中地衣芽孢杆菌、浸麻芽孢杆菌及混合菌剂2的升温作用更为明显。冬季堆肥试验，以2个市售发酵菌剂和不接种空白处理为对照，比较了接种9个单一菌剂及它们复合菌剂的堆肥升温效果，筛选出肥堆升温和促腐效果达到或超过酵素菌的菌株6个：地衣芽孢杆菌、枯草芽孢杆菌、浸麻芽孢杆菌、里氏木霉、CMC-W70-S、CS-R30-P。春季堆肥试验结果表明，采用6个菌株制备的混合菌剂接种处理的肥堆升温、保氮、pH和有机肥全氮磷钾含量及有机质含量达到或超过市售发酵菌剂的接种效果，表明本研究开发的利用有机废液生产的发酵菌剂在禽畜粪高温堆肥中具有实际应用价值。
(4)通过番茄基质培盆栽试验、大棚滴灌试验和西瓜田间试验，研究了改性味精废液作为番茄基质栽培营养液的可行性以及采用有机废液制备的联合固氮菌W12和地衣芽孢杆菌等微生物制剂对西瓜、番茄的接种效应。①番茄基质培盆栽试验结果表明，浇施味精废液处理可显著增加番茄株高、叶片数和茎粗，促进其营养生长，并使番茄提早开花，有利于生殖生长，尽管始花期恰逢连阴雨天气影响初期花的挂果，但产量仍略高于完全营养液处理。而且，栽培基质的电导率与浇施完全营养液处理无显著差异。②味精废液与W12菌剂联用促生效果基质培盆栽试验结果表明，番茄茎、叶的全氮量表现为接种W12处理极显著高于不接菌对照，浇味精废液处理极显著高于其它两个营养液，且番茄茎全氮含量在基质接种方式与不同营养液间的交互作用极显著，说明浇施味精废液不仅可直接为番茄植株提供营养，还可以为W12提供丰富的营养源。浇施味精废液的番茄产量显著高于完全营养液处理，而且生长后期的下位叶黄化程度明显比后者减轻。虽然浇施味精废液的栽培基质pH略低于完全营养液处理，基质的电导率略高于后者，但均在番茄生长安全pH和电导率范围内。③樱桃番茄基质培大棚滴灌味精废液试验结果，进一步证实了味精废液能改善番茄叶片的氮素营养，显著提高其叶绿素和类胡萝卜素含量。滴灌味精废液的番茄功能叶抗衰老相关因子SOD、MDA、POD和常规营养液无显著差异，却显著提高叶片可溶性蛋白含量，说明有利于提高番茄叶片的抗逆性。滴灌味精废液不仅取得与滴灌常规无机营养液相同的产量，而且果实总氨基酸增加30％，总糖度及糖酸比显著提高，总酸度明显降低，明显改善番茄果实的鲜食风味和营养品质。此外，微生物菌剂对西瓜接种田间试验结果表明，接种采用味精废液制备的地衣芽孢杆菌菌剂能使西瓜产量有所提高。综上所述，经过改性的味精废液可以作为基质培番茄营养液，且能与联合使用的功能微生物菌剂产生显著的正交互作用，提高功能微生物接种效果。该项技术已获国家发明专利授权(ZL200510049612．0)。
(5)为探讨土壤化学和生物学性状与外源固氮菌对土著菌竞争力的关系，采用联合固氮菌W12的利福平诱导抗性菌株W12-R，设计了W12-R在3个熟化度和肥力水平差异悬殊的土壤浸提液中与土著菌的竞争生长试验。结果表明，W12-R在3个土壤浸提液中单独培养时生长量无显著差异，但与土著菌共培养时，W12-R的生长受到了不同程度的抑制，其中以在有效养分含量最低的洪合土壤浸提液中受到的竞争抑制最大。补充适量碳源可不同程度削弱这种抑制，其中在洪合土壤浸提液中最为突出，使W12-R生长量回复到纯培养水平。比较在无氮培养基中W12单独以及分别与各供试土壤土著菌共培养系统中测得的固氮酶活性，发现共培养系统的固氮酶活性相对于纯培养系统大幅下降，而且三个土壤的共培养体系的固氮酶活性差异极显著(p=0.0001)，其中以总氮、有机质、微生物量最高的新埭土壤共培养系统最低，而各项土壤特性指标相对较低的洪合土壤共培养系统最高。可见，外源固氮菌W12与土著菌共存时，土著微生物不仅对其生长造成营养竞争抑制，对其固氮酶活性也产生了显著的影响。进一步分析土壤NH_4~+-N含量、微生物量碳、微生物量氮、TOC以及土著细菌、真菌和放线菌数量与共培养系统固氮酶活性的关系，结果表明土壤NH_4~+-N含量和土著微生物数量越高，W12与土著菌共培养系统的固氮酶活性降低越多。由上述结果可见，对微生物量较少，肥力较低，特别是NH_4~+-N含量较低的土壤接种外源固氮菌W12，并利用固、液有机废弃物补充适当的营养源，将有利于提高其接种效果。
(6)多元统计分析结果表明，土壤土著菌固氮酶活性NAS仅与13项土壤性状指标中的土壤有效态Cd呈显著偏负相关；外源菌W12与土著菌共培养系统的固氮酶活性NA(S+W12)及W12接种效应估计值NAD(NA(S+W12)-NAS)分别与土壤NH_4~+-N、有效态Cr含量呈显著的负偏相关和正偏相关；NA(S+W12)与NAS呈显著的正相关，而NAD却与NAS呈显著的负相关，表明接种W12后共培养系统的总固氮量有一部分是来自土著固氮菌的贡献，但较高NAS却不利于提高外源固氮菌W12的接种效应。通过系统聚类分析将27个供试土样分为外源固氮菌W12接种效应明显不同的3个类型，其中，第一类由于接种效应正相关因子土壤有效Cr水平较高，而负相关因子NH_4~+-N、NAS居中等水平，因而表现为正种效应；第二类因土壤有效态Cd过高，表现为负接种效应；第三类可能由于NAS过高而具有负接种效应。根据外源固氮菌W12接种效应估计值NAD与其正相关因子土壤有效态Cr含量及负相关因子土壤NH_4~+-N含量的关系曲线，初步发现在供试土壤范围内，两者对外源固氮菌W12接种效应产生胁迫的临界值分别为2μg kg~(-1)和20mg kg~(-1)，即土壤有效态Cr含量低于2μg kg~(-1)和／或土壤NH_4~+-N含量高于20mg kg~(-1)将抑制外源固氮菌W12的接种效应发挥。
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 SO_4~(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 L_9(3~4) orthogonal design experiments for 3 factors, e.g. MGW, bean products wastewater (BPW) and cane sugar. Based on L_(16)(2~(15)) 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.①The 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.②The 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.③Results 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 NH_4~+-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 NH_4~+-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 NH_4~+-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 NH_4~+-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 NH_4~+-N content, the stress threshold of soil available Cr and soil NH_4~+-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 NH_4~+-N content higher than 20mg kg~(-1).
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