摘要
铜是生物正常生长发育所必需的微量元素,但过量的铜会干扰动植物和人的正常生理功能,造成植物生长缓慢甚至死亡且危害人类健康,因此铜污染修复具有重要的现实意义。木霉属真菌可通过分离培养获得纯培养、易于筛选,生物量大且适应酸性环境,且具有有重金属抗性或促进植物生长的功能,具有用于铜污染土壤修复的优势。鉴此,本研究以一株经筛选的里氏木霉FS10-C为研究对象,探讨其对铜污染土壤的强化修复效应和机理。主要研究内容和结果如下：
1)通过不同温度、湿度、pH和光照下里氏木霉FS10-C孢子萌发、菌丝生长及产孢情况探讨里氏木霉FS10-C的环境适应性,并初步评估了里氏木霉FS10-C的生物安全性,以评价其环境应用前提。结果表明,里氏木霉FS10-C在10-40℃和相对湿度60%～100%时均可进行孢子萌发和菌丝生长；在20～35℃时可产孢；在铜污染导致的偏酸性环境中(pH4-7)生长繁殖旺盛；光照利于产孢但对孢子萌发和菌丝生长影响不大。同时,里氏木霉FS10-C及其代谢产物无皮肤刺激性、无急性皮肤毒性不产生溶血效应。可见,里氏木霉FS10-C环境适应性广且对人畜无害,初步具备应用于环境铜污染修复前提条件。
2)通过摇瓶试验研究铜胁迫条件对里氏木霉FS10-C生长发育和积累铜的影响、对里氏木霉FS10-C成熟细胞形态及吸附铜的影响及对里氏木霉FS10-C生理特性的影响等,同时研究了里氏木霉FS10-C产铁载体能力,探讨该株木霉的铜胁迫响应及其抗性机制。结果表明：(1)里氏木霉FS10-C在Cu2+浓度不大于600mg/L的溶液中均可生长发育,且在含100、200和400mg/L Cu2+的溶液中铜积累率分别可达50.6%、30.3%和4.7%。SEM显示,在含100mg/L Cu2+溶液中,成熟里氏木霉FS10-C细胞形态无明显改变,400mg/L时菌体结构仍完整；EDX元素分析显示,在含100和400mg/L Cu2+的溶液中,里氏木霉FS10-C细胞铜的平均质量分数分别为18.50%和30.40%,且菌丝大于孢子；(2)里氏木霉FS10-C的铜抗性机制与细胞表面含氮基团和磷脂的作用、可溶性蛋白和GSH含量的增加及铁载体的分泌有关。可见,里氏木霉FS10-C是一株高铜抗性真菌,可通过菌体成分及生理生化调节机制积累和吸附铜,减少环境中的铜。
3)采用正交试验筛选最佳固体里氏木霉FS10-C制剂发酵方法。通过盆栽试验研究里氏木霉FS10-C木霉制剂对外源铜污染土壤海州香薷生长和铜吸收能力及对土壤质量的影响。结果表明：(1)里氏木霉FS10-C以苜蓿粉为固体发酵基质,在适宜发酵条件下可制成孢子含量较高的木霉制剂；(2)施加该木霉制剂后,除400mg/kg外源铜浓度土壤上地上部干重外,海州香薷生物量在各外源铜浓度土壤上均呈增加趋势；各外源铜浓度土壤NH4OAc提取态铜含量均显著高于PD培养基组(P<0.05),且除l00mg/kg外源铜土壤上地上部外,海州香薷铜积累量均呈增加趋势,总积累量在100和400mg/kg外源铜土壤上均显著增加(P<0.05)；(3)该木霉制剂显著提高了外源铜土壤上土壤速效磷含量、脲酶、脱氢酶和磷酸酶等土壤酶活性、土壤生物量碳含量及土壤pH,从而提高土壤质量。可见,里氏木霉FS10-C制剂可通过提高海州香薷生物量和活化土壤铜有效性增加其对海州香薷对铜的吸收能力,同时可显著提高土壤质量,具有土壤修复剂和微生物肥料双重功效。
4)通过研究里氏木霉FS10-C对海州香薷的促生作用、促生生物学特性、对海州香薷抗铜胁迫能力(铜抗性)的影响及里氏木霉FS10-C对土壤铜有效性的影响,探讨了里氏木霉FS10-C对海州香薷修复铜污染土壤强化作用的机理。结果表明：(1)里氏木霉FS10-C可产铁载体、ACC脱氨酶且具有较强的溶磷能力等促生生物学活性；(2)在无铜胁迫时,里氏木霉FS10-C可显著提高海州香薷萌发率,促进幼苗根系发育；(3)铜胁迫环境下,里氏木霉FS10-C亦可显著促进海州香薷种子萌发、提高生物量和根系活力,降低MDA含量、提高GSH含量及提高POD、SOD和CAT活性(P<0.05)；(4)里氏木霉FS10-C生长发育的过程中降低培养基pH,发酵液有较强的浸提土壤铜的作用,提示里氏木霉FS10-C在代谢过程中释放酸性物质提高土壤铜有效性。可见,里氏木霉FS10-C通过促生活性和增强铜抗性促进铜胁迫下海州香薷的生长发育,通过产生酸性产物等提高土壤铜有效性,从而强化修复铜污染土壤。
综合以上研究认为,里氏木霉FS10-C是一株颇具研究潜力的铜污染生物修复菌,其木霉制剂可在铜污染土壤植物修复中发挥安全有效的强化作用。
Copper is an essential trace element that is vital to the health of all living things. But excess copper accumulation in plants, animals and human being will disturb their normal physiological processes, lead to stunted growth or even death in plants, and worse enough, risk factors of animals and our human beings. Therefore, remediation of copper contamination is of great importance at present. Trichoderma sp. can be easily screened out by isolation and culturing methods to cost-effectively obtained their pure culture. In addition, Trichoderma sp. has larger production of biomass, and can grow well in low pH besides its capacity of heavy metal resistance or promoting plant growth. Therefore, it could be as a candidate with its great advantage for remediation of copper contamination. A copper resistant Trichoderma reesei strain FS10-C was studied in the present study, to investigate its effects and mechanisms in bioremediation of copper contaminated soil. The important research scope and results are summarized as follows:
1) Trichoderma reesei FS10-C was evaluated for its environmental adaptability through verifying of the spore germination, hyphal growth and sporulation in diverse conditions of temperature, humidity, pH value and light. In addition, bio-safety assessment was conducted to evaluate the perspective application of Trichoderma reesei FS10-C in remediation of copper pollution. The results showed that spore germination and hyphal growth of Trichoderma reesei FS10-C were possible at temperature of10-40℃and in relative humidity of60-100%, and sporulation occurred favorably at20-35℃. Trichoderma reesei FS10-C grew vigorously in acid-biased environment (pH4-7) due to copper contamination. Light stimulated sporulation sporulation of Trichoderma reesei FS10-C, but had little effect on spore germination and hyphal growth. No toxicity effects of skin irritation, skin toxicity, eye irritation and hemolysis were observed in both Trichoderma reesei FS10-C and its metabolites. These findings show that Trichoderma reesei FS10-C has wider environmental suitability, yet no health risk factors for humans and animals. It suggested that Trichoderma reesei FS10-C is a safe candidate for bio-remediation of copper contamination.
2) Shake Flask tests were performed to observe variations of Trichoderma reesei FS10-C under copper stress, such as growth and development while copper accumulation, morphology and element of adult cell while copper absorption, and physiological-biochemical characteristic. Besides, capacity of siderophore production was verified to study responses and mechanisms of copper resistance of Trichoderma reesei FS10-C. The results showed that (1) the spores of Trichoderma reesei FS10-C were well grown and developed in aqueous solutions with Cu(II) concentration below600mg/L. Copper accumulation rates of Trichoderma reesei FS10-C under100,200and400mg/L Cu(II) concentrations were50.6%,30.3%and4.7%, respectively. The SEM showed no morphological variation in Trichoderma reesei FS10-C at100mg/L Cu(II) and mostly complete cell structure at400mg/L Cu(II). EDX results showed that weight percentage of copper in Trichoderma reesei FS10-C was18.50%and30.40%at100and400mg/L Cu(II), respectively, and biosorption capacity of mycelium was higher than that of spore;(2) mechanisms of copper resistant and removal effect of Trichoderma reesei FS10-C were related to increase of soluble protein and GSH contents, siderophore production, nitrogen-containing group and phospholipid of cells. It suggested that Trichoderma reesei FS10-C is a fungal strain with higher copper resistance, and it can remove copper by bioaccumulation and biosorption by cell composition and physiological and biochemical regulating mechanisms.
3) Spores of Trichoderma reesei FS10-C were made into Trichoderma sp. preparation by solid-state fermentation with condition screened by orthogonal tests. And a pot experiment of Elsholtzia splendens in soils spiked with copper was carried out to investigate effects of the preparation on growth and Cu accumulation of the plant and on soil quantity. The results indicated that (1) spores of Trichoderma reesei FS10-C can grow and develop in alfalfa meal as solid matrix and made into Trichoderma sp. preparation with high spore density;(2)the plant biomass of Elsholtzia splendens showed an increasing trend in soils with all spiked Cu concentrations after applying of the preparation, except for shoot dry weight in soils with spiked Cu levels of400mg/kg. The preparation increased NH4OAc-extractable Cu in all Cu contaminated soils significantly in comparison with that in the control (P＜0.05), and also led to an increasing trend in Cu accumulations of all plants, except for that in shoot in soils with external Cu level of100mg/kg. Total Cu accumulations were significantly enhanced in soils with external Cu levels of100and400mg/kg (P＜0.05);(3)the Trichoderma sp. preparation improved soil quantity by enhancing available P content, activity of dehydrogenase, urease and phosphatase, microbial biomass C content and pH value of Cu contaminated soil. The findings suggested that this Trichoderma reesei FS10-C preparation could enhance Cu accumulation by promoting growth of Elsholtzia splendens and increasing Cu phytoavailability in soil. It can be both bioremediation agent and microbial fertilizer for soil, and could be a potential agent for bioremediation of copper contaminated soil.
4) The mechanisms of augmentation effect of Trichoderma reesei FS10-C on remediation of Cu contaminated soil with Elsholtzia splendens were investigated, such as plant growth promoting effect on Elsholtzia splendens, biological characteristics of plant growth promoting, enhancement of Cu resistance of Elsholtzia splendens and enhancement Cu. availability of soil.The results showed that (1) Trichoderma reesei FS10-C was able to produce ACC deaminase, siderophore, and have the ability of phosphate solubilization;(2) Trichoderma reesei FS10-C had significantly promoted seed germination of Elsholtzia splendens and root development of the seedling in condition of copper stress free;(3)even in environment of copper stress, Trichoderma reesei FS10-C were capable of promoting seed germination and enhancing biomass, improving root activity, decreasing MDA content and increasing POD, SOD and CAT activities of Elsholtzia splendens(P<0.05).(4) Trichoderma reesei FS10-C had reduced pH value of the culture media during its development process. The fermentation broth also had higher ability of extracting Cu from soils. It proved that Trichoderma reesei FS10-C may release certain acidic materials that were able to accelerate extraction of Cu from soils to eventually enhance Cu availability of soil. These findings suggested that Trichoderma reesei FS10-C could promote growth and development of the plant in Cu contaminates soil due to its biological activity of plant growth promoting and capacity of copper resistance enhancement, and it may enhance Cu. availability of soils due to producing some acid materials.
In summary, copper resistant Trichoderma reesei FS10-C can be a potential candidate for bioremediation of copper pollution. Trichoderma reesei FS10-C preparation could play safe and effective role in phytoremediation of Cu contaminated soil.
引文
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