摘要
菌丝球由于具有一定的机械强度、多孔网状、表面光滑均匀的独特结构特征,因此被作为一种新型的生物质载体而备受关注。以菌丝球作为固定化微生物的新型生物质载体,采用混合菌种固定化技术,形成固定化菌丝球,建立多菌种共生的微生态环境,使各种固定化微生物协同发挥作用,极大地扩展了微生物在环境生物技术领域的应用范围。本研究以本实验室筛选获得的两株具有木质纤维素降解能力的海洋真菌为基础,构建了一种新型的双菌种固定化体系---固定化菌丝球,并对该体系在造纸废水处理中的应用进行初探,取得了如下的结果：
首先从我国浙江东部沿海海域采集到的样品中初步分离出8株具有纤维素降解活力的真菌,筛选出25株具有木质素降解活力的真菌。经过进一步复筛,分别获得一株能产较高纤维素酶的菌株和一株能产较高漆酶的菌株。经菌落形态鉴定以及rDNA-ITS序列鉴定,能产较高纤维素酶的海洋真菌鉴定为微紫青霉属(Penicillium janthinellum),并命名为P1；另一株产较高漆酶的海洋真菌鉴定为内生拟盘多毛孢菌属(Pestalotiopsis sp.),并命名为J63。
第二,鉴于文献中有关内生拟盘多毛孢菌产漆酶的研究报道很少,因此本文对海洋内生拟盘多毛孢菌J63产漆酶进行了较为全面的发酵性能考察。重点考察了培养基的组成(碳源、氮源的种类,不同铜离子浓度以及铜离子的加入时间、不同盐浓度)、培养条件(温度,起始pH)以及外加诱导剂等对产漆酶的影响。实验结果表明在最优的发酵条件下,可得到漆酶酶活5719.7U/L,要高于许多真菌产漆酶的能力。而且该菌株具有广域耐盐性,因此是一株非常具有竞争力的产漆酶菌株。
第三,为了充分利用废弃资源,重点选用了7种农业废弃物(稻草粉、麸皮、水葫芦、甘蔗渣、豆荚粉、玉米芯和柚子皮)为底物进行固体发酵生产漆酶,其中水葫芦更是被称为全球十大害草之一。实验结果表明J63菌株利用农业废弃物生产漆酶的潜力巨大,其中以稻草粉发酵产漆酶的能力最强,其次为水葫芦,实现了环境友好型高效低成本生产漆酶。
第四,通过紫外诱变和离子注入诱变对J63菌株进行菌种改造,经过初筛和复筛,得到两株正突变菌株A6和B21,其第5天产酶酶活比出发菌株J63分别提高了38.4%和35.5%。而且,菌株A6在耐热性与耐盐性上都有显著提高,酶活分别为出发菌株J63的5-6倍,这将为菌株A6在未来的实际工业应用中创造非常有利的条件。
第五,海洋真菌微紫青霉菌P1在一定的液体培养条件下能够形成具有空间网状结构的菌丝球。本研究以菌丝球为载体,通过包埋固定化方法将海洋真菌内生拟盘多毛孢菌J63孢子进行固定,构建了一种新型双菌种全细胞固定化体系。此体系既具有纤维素降解能力,同时还具有木质素降解能力。通过正交试验设计,得到了最优的固定化条件：P1菌丝球的量为10g(湿重)；J63孢子浓度为2×109个/mL；共固定化时间为2d。
第六,考察了固定化菌丝球对造纸废水进行生物处理的影响效果,重点研究了不同浓度外加碳源、不同浓度外加氮源、固定化菌丝球用量、温度和初始pH的影响。实验表明,固定化菌丝球可以非常有效地对造纸废水进行生物处理,在10h的处理时间内,生物降解率达到99%以上。而且,经过6批次的循环处理使用,生物降解率仍高达96.4%。表明固定化菌丝球可以较长时间地维持活性,持续、高效地处理废水。在废水处理的过程中,发现菌丝球出现再生长现象。经过6批次废水处理后的固定化菌丝球直径有3.7mm,是未经废水处理的固定化菌丝球直径的1.6倍。经固定化菌丝球处理过的废水由原本呈深绿色、浑浊不清、含有大量细微的不溶性纤维样物质、有恶臭的水变成了无色、澄清、无异味的水。
最后,将固定化菌丝球和P1菌丝球分别应用于处理模拟染料废水的脱色研究。在脱色培养10d后,两者都能有效地对染料进行脱色,固定化菌丝球的脱色率比P1菌丝球的脱色率高出近20%。再一次证明了,这种新型的共固定化体系在实际的工业应用中比单纯的菌丝球生物处理具有更加优越的性能,应用范围更加广泛。
It is noteworthy that mycelia pellet possesses a unique structural characteristic, poly-porous network and high surface area, which has been noticeably exploited as a new kind of biological carrier. Using the mycelium pellet as a biological carrier in the immobilized microorganisms, the resulting immobilized pellet has been formed by adopting the "mixed bacteria self-immobilization technology", which establishes a micro-ecology environment made up of mixed bacteria group in a small community, in order that letting all kinds of immobilized microorganisms play their roles together. This sort of immobilized pellet has greatly expanded its appliance in the wastewater treatment. In our research, two strains of marine-derived fungi, isolated by ourselves, which can secrete lignocelluloses, were utilized to construct a novel two-specie whole-cell immobilization system, then applied this system into wastewater treatment.
Firstly, we isolated and screened25strains of fungi which are of the capability of degradation of lignin, and8strains of fungi capable to degrade cellulose from sediment, sea-grass and sea-mud samples collected from East China Sea. After further round of screen, a pure culture which secreted higher laccase and another pure culture which produced higher cellulase were picked out. Through the identification of colony morphology and rDNA-ITS, the strain which secreted higher laccase was identified as Pestalotiopsis sp. and named it J63; another strain produced higher cellulase was identified as Penieillium janthinellum, named it P1.
Secondly, to the best of our knowledge production of laccase by Pestalotiopsis sp. was rarely documented. So, this work primarily investigated comprehensively the fermentation properties of marine-derived fungus Pestalotiopsis sp. J63, mainly focusing on the effect of production of laccase by the components of medium (including carbon source, nitrogen source, Cu2+concentration, different salinities etc.), cultivation condition (including temperature, initial pH) and extra-added inducers. Results showed that under the optimum fermentation condition the capability of producing laccase can reach at5719.7U/L, which is superior to other fungi's capability. Additionally, strain J63is a halotolerant microorganism. Therefore, strain J63is considered as a pretty competitive strain to produce a large amount of laccase.
Thirdly, in order to fully explore the waste resources, seven agricultural wastes, containing rice straw powder, wheat bran, bean-pod powder, sugarcane bagasse, corn cob powder, water hyacinth and the peel of pomelo, were picked out as substrate to produce laccase under solid-state fermentation. Among them, Water hyacinth is viewed as one of the most harmful grasses in the world. So, there is a far-reaching influence if it can turn wastes into wealth. Results demonstrated that the potential of utilizing agricultural wastes to produce laccase is fairly tremendous, in which the capability of production of laccase by utilization of rice straw powder excelled others, reaching10700U/g. The runner-up is water hyacinth,7593.3U/g. These achievements met the goal of production of laccase in a manner of environmental-friendly means.
Fourthly, through UV-mutagenesis and ion implantation mutagenesis for mutation breeding, two of positive mutant strains, A6and B21, were screened after two rounds of screening procedures. Their laccase ability increased38.4%and35.5%in comparison with parent strain J63in the5th day of fermentation respectively. Furthermore, strain A6improved notably either in thermostability or in halotolerancy, whose laccase enzyme activity is5-6fold as high as its parent strain J63. This is very beneficial to apply in practical industrial sectors.
Fifthly, marine-derived fungus Penicillium janthinellum P1can form mycelia pellets in a specific liquid culture. Mycelia pellet has been exploited as biological carrier for whole-cell immobilization due to its unique structural characteristic---polyporous and high specific area. This study constructed a novel two-specie whole-cell immobilization system which using P1mycelia pellets as carrier, which was achieved simply by inoculating the marine-derived fungus Pestalotiopsis sp. J63spores into culture medium containing another fungus Penicillium janthinellum P1pre-grown mycelia pellets. This so-constructed immobilization system possesses the ability not only to degrade lignin but also to degrade cellulose. Optimum conditions of immobilized procedure for maximum biodegradation capacity were determined using orthogonal design at biomass of P1pellets,10g (wet weight); concentration of J63spore, 2×109/mL; and immobilization time,2d.
Sixthly, our work investigated the effect of biological wastewater treatment by utilization of immobilized pellets, mainly concentrating on different concentrations of extra carbon source and nitrogen source, different dosages of immobilized pellets, temperature and initial pH. Results demonstrated that immobilized pellets can be fairly effective to biodegrade paper mill effluent in a ten-hour batch run. Biodegradation capacity exceeded99%. Furthermore, biodegradation capacity still retained96.4%after6batches of consecutive treatment. This exhibited that immobilized pellets are capable to maintain very active for dealing with effluent efficiently and persistently for a longer time. During wastewater treatment, re-growth phenomenon of immobilized pellets occurred. The average diameter of immobilized pellets treated by6batches of semi-successive effluent treatment, was3.7mm, which is1.6fold as large as those untreated immobilized pellets. After treatment, the wastewater, originally feculent, turned out to be clarified and odourless, without visible particles.
At last, immobilized pellets and P1mycelia pellets were applied to treat simulated dye solution Azure B respectively. It was revealed that both P1mycelia pellets and immobilized pellets can effectively decolorize Azure B. However, the decolorization capability of immobilized pellets improved notably, by26%, compared with mycelia pellets during a10-day treatment. This further confirmed that this co-immobilization system was more advantageous than single mycelia pellets in industrial application.
引文
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