高效菌降解造纸黑液木质素的特性与机理研究
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摘要
造纸工业是对环境污染非常严重的行业之一，治理造纸废水的关键是对木质素的有效处理。本文通过大量的实验研究工作，在初步探索造纸黑液酸析木素新助剂及其效果的基础上，首次系统地对木素降解高效菌的分离与生长繁殖特性、高效菌酶活性与木素降解性能、生物强化因子综合实验及实际模拟试验等进行了深入研究，为提高造纸黑液木素的处理效果提供理论依据。
本研究通过大量的实验，获得了改进酸析木素法的新助剂，与常规酸析法相比，该助剂使用后具有较好的COD_(Cr)和色度去除率，析出的木素颗粒上浮且含水率低等优点。当pH值为3.0时，黑液COD_(Cr)去除率最高可达68％，色度去除率为92％以上，浮渣含水率最低仅为81％，并且pH在4～4.5时仍有类似的去除木素效果，这些有助于克服常规法中耗酸量大、泥水分离困难、需要加热设备、污泥后续处理困难等缺点。实验证明，Ⅰ号助剂的作用效果最好。
从119个菌株中分离筛选出了6株细菌和5株霉菌，它们可以在含有木质素的培养基上良好生长：细菌分类学鉴定结果为X1、X2和X3：假单胞菌属，X4和X5：黄单胞杆菌属，X6：枝动杆菌属；对6株细菌降解木质素性能进行了初步的研究，选定X2和X4为实验用菌种，两株菌适宜的生长温度为30℃，最适初始pH值为7；经紫外线照射诱变法处理，可使它们产生的三种木素分解酶活性均有明显的提高，细菌增长速率比诱变前明显提高。
对分离出的高效菌种的特性进行了较为详细的研究，主要研究结果是：110转／min振荡培养有利于菌种的生长繁殖；通过对多种共代谢初级碳源和氮源的效能比较，确定蔗糖为合适的共代谢初级碳源，硝酸铵为合适的氮源；向原降解培养基中加入10mg／L的Mn~(2+)离子，0.1mg／L的Cu~(2+)离子，0.25mg／L的Fe~(2+)离子和10mg／L的Ca~(2+)离子，会大大提高木质素的去除率；综合培养结果表明，加入共基质蔗糖的浓度为1g／L，硝酸铵浓度为1.5g／L和相应的无机离子，且菌种比例为X2：X4=1：1时，效果最好。
西安建筑科技大学博士学位论文
根据酶活性测定结果和降解中间产物色谱一质谱联机分析，认为高效茵降解木质素的机理
是，在综合强化因于作用下，促进木质素降解酶活性提高，强化了木质素的胞外和胞内降解反
应。
采用投加生物强化因子的生物强化处理系统，对造纸废水进行连续的模拟运行实验，也获
得了很好的效果，这些强化因子包括木质素降解高效菌种、共代谢初级碳源、氮源以及多种无
机离于等；综合强化因子作用下的COD最高去除率达75．3％，平均COD去除率在74％以上，
COD值从进水的1000呗左右降到了约260m叭，SS的去除率也在90％以上。
Paper industry is one of the industries that cause serious environmental pollution. The key of paper industry sewage treatment is the efficient treatment of lignin. By a host of experiments, On the basis of exploration of new assistant reagent and its effect for paper industry black liquor, a detailed study had been done on separation of efficient lignin-degrading bacteria, their growth, reproduction, enzyme activity and lignin-degrading capacity, and synthetic experiments of bioaugmentation factors and practical simulated experiments, which offer theoretical basis to improve removal effect of lignin in paper industry black liquor.
By a host of experiments, the study lias got new assistant reagents for modified acid precipitation process. Compared with the conventional, process using of the reagents has such advantages as higher removal ratio of CODcr and chroma, and lower water content of the deposited lignin particles. When the pH value is 3.0, the highest CODcr removal ratio can reach 68%, the chroma removal ratio over 92%, and lowest water content of scum is 81 %. The similar removal effect of lignin occurs when the pH is controlled between 4 and 4.5, which is helpful to conquer the disadvantages of large amount of acid consumption, hard to dehydrating, necessity of heating equipments, and difficult treatment of sludge. The result showed that the NO. 1 assistant reagent has the best effect.
Six bacteria and five mildews have been separated from 119 strains, wliich grew well on the culture media contained lignin. The result of taxonomic identification is that XI, X2 and X3 belong to Pseudomonas, X4 and X5 belong to Xanihomonas, X6 belongs to Mycoplana. A preliminary study has been carried out on the capacity of lignin degradation of the 6strains of bacteria, and strains X2 and X4 were chose to carry out the experiments. The most favorable temperature for the 2 strains is 30癈, and the pH is 7; After being treated by ultraviolet rays, activity of all the three kinds of lignin-degrading enzyme produced by the strains can be increased greatly, the bacteria increasing rate is remarkably improved.
Detailed study has been carried out on specific property of the separated efficient bacteria, the chief results are that shaking culture at 110r/min is favorable for the growth and reproduction of the bacteria; After comparing the effects among kinds of primary carbon and nitrogen sources, sucrose is determined as the suitable primary source for co metabolizing, and ammonium nitrate as the nitrogen source; the removal ratio of lignin can be greatly unproved after adding 10mg/l manganese ion, 0.lmg/1 copper ion, 0.25mg/l ferrous ion and 10mg/1 calcium ion into the culture media; Synthetically cultivating results show that the best effect occurs when the concentration of sucrose is 1 mg/1, ammonium nitrate is 1.5mg/l with corresponding inorganic ion, and the proportion of bacteria isX2:X4 equals 1:1.
According to the determination of enzyme activity and chromatogram- mass spectrum analysis of intermediate, the mechanism of lignin-degrading by efficient bacteria is that the synthetically bioaugmentation factors improve the activity of lignin-degrading enzymes, which augment die lignin-degrading reaction in outer cell and inner cell.
A ideal effect has been got as well when continuous simulated experiments were done to treat paper industry sewage in a bioaugmentation system added with bioaugmentation factors, which included effect lignin-degrading bacteria, primary carbon for co metabolizing, nitrogen sources and kinds of inorganic ions; under the function of synthetic bioaugmentation factors, the highest COD removal ratio is 75.3%, the average is over 74%, the COD value descend from l000mg/L in influent to about 260mg/L in effluent, and the removal ratio of scum is over 90%.
本研究通过大量的实验，获得了改进酸析木素法的新助剂，与常规酸析法相比，该助剂使用后具有较好的COD_(Cr)和色度去除率，析出的木素颗粒上浮且含水率低等优点。当pH值为3.0时，黑液COD_(Cr)去除率最高可达68％，色度去除率为92％以上，浮渣含水率最低仅为81％，并且pH在4～4.5时仍有类似的去除木素效果，这些有助于克服常规法中耗酸量大、泥水分离困难、需要加热设备、污泥后续处理困难等缺点。实验证明，Ⅰ号助剂的作用效果最好。
从119个菌株中分离筛选出了6株细菌和5株霉菌，它们可以在含有木质素的培养基上良好生长：细菌分类学鉴定结果为X1、X2和X3：假单胞菌属，X4和X5：黄单胞杆菌属，X6：枝动杆菌属；对6株细菌降解木质素性能进行了初步的研究，选定X2和X4为实验用菌种，两株菌适宜的生长温度为30℃，最适初始pH值为7；经紫外线照射诱变法处理，可使它们产生的三种木素分解酶活性均有明显的提高，细菌增长速率比诱变前明显提高。
对分离出的高效菌种的特性进行了较为详细的研究，主要研究结果是：110转／min振荡培养有利于菌种的生长繁殖；通过对多种共代谢初级碳源和氮源的效能比较，确定蔗糖为合适的共代谢初级碳源，硝酸铵为合适的氮源；向原降解培养基中加入10mg／L的Mn~(2+)离子，0.1mg／L的Cu~(2+)离子，0.25mg／L的Fe~(2+)离子和10mg／L的Ca~(2+)离子，会大大提高木质素的去除率；综合培养结果表明，加入共基质蔗糖的浓度为1g／L，硝酸铵浓度为1.5g／L和相应的无机离子，且菌种比例为X2：X4=1：1时，效果最好。
西安建筑科技大学博士学位论文
根据酶活性测定结果和降解中间产物色谱一质谱联机分析，认为高效茵降解木质素的机理
是，在综合强化因于作用下，促进木质素降解酶活性提高，强化了木质素的胞外和胞内降解反
应。
采用投加生物强化因子的生物强化处理系统，对造纸废水进行连续的模拟运行实验，也获
得了很好的效果，这些强化因子包括木质素降解高效菌种、共代谢初级碳源、氮源以及多种无
机离于等；综合强化因子作用下的COD最高去除率达75．3％，平均COD去除率在74％以上，
COD值从进水的1000呗左右降到了约260m叭，SS的去除率也在90％以上。
Paper industry is one of the industries that cause serious environmental pollution. The key of paper industry sewage treatment is the efficient treatment of lignin. By a host of experiments, On the basis of exploration of new assistant reagent and its effect for paper industry black liquor, a detailed study had been done on separation of efficient lignin-degrading bacteria, their growth, reproduction, enzyme activity and lignin-degrading capacity, and synthetic experiments of bioaugmentation factors and practical simulated experiments, which offer theoretical basis to improve removal effect of lignin in paper industry black liquor.
By a host of experiments, the study lias got new assistant reagents for modified acid precipitation process. Compared with the conventional, process using of the reagents has such advantages as higher removal ratio of CODcr and chroma, and lower water content of the deposited lignin particles. When the pH value is 3.0, the highest CODcr removal ratio can reach 68%, the chroma removal ratio over 92%, and lowest water content of scum is 81 %. The similar removal effect of lignin occurs when the pH is controlled between 4 and 4.5, which is helpful to conquer the disadvantages of large amount of acid consumption, hard to dehydrating, necessity of heating equipments, and difficult treatment of sludge. The result showed that the NO. 1 assistant reagent has the best effect.
Six bacteria and five mildews have been separated from 119 strains, wliich grew well on the culture media contained lignin. The result of taxonomic identification is that XI, X2 and X3 belong to Pseudomonas, X4 and X5 belong to Xanihomonas, X6 belongs to Mycoplana. A preliminary study has been carried out on the capacity of lignin degradation of the 6strains of bacteria, and strains X2 and X4 were chose to carry out the experiments. The most favorable temperature for the 2 strains is 30癈, and the pH is 7; After being treated by ultraviolet rays, activity of all the three kinds of lignin-degrading enzyme produced by the strains can be increased greatly, the bacteria increasing rate is remarkably improved.
Detailed study has been carried out on specific property of the separated efficient bacteria, the chief results are that shaking culture at 110r/min is favorable for the growth and reproduction of the bacteria; After comparing the effects among kinds of primary carbon and nitrogen sources, sucrose is determined as the suitable primary source for co metabolizing, and ammonium nitrate as the nitrogen source; the removal ratio of lignin can be greatly unproved after adding 10mg/l manganese ion, 0.lmg/1 copper ion, 0.25mg/l ferrous ion and 10mg/1 calcium ion into the culture media; Synthetically cultivating results show that the best effect occurs when the concentration of sucrose is 1 mg/1, ammonium nitrate is 1.5mg/l with corresponding inorganic ion, and the proportion of bacteria isX2:X4 equals 1:1.
According to the determination of enzyme activity and chromatogram- mass spectrum analysis of intermediate, the mechanism of lignin-degrading by efficient bacteria is that the synthetically bioaugmentation factors improve the activity of lignin-degrading enzymes, which augment die lignin-degrading reaction in outer cell and inner cell.
A ideal effect has been got as well when continuous simulated experiments were done to treat paper industry sewage in a bioaugmentation system added with bioaugmentation factors, which included effect lignin-degrading bacteria, primary carbon for co metabolizing, nitrogen sources and kinds of inorganic ions; under the function of synthetic bioaugmentation factors, the highest COD removal ratio is 75.3%, the average is over 74%, the COD value descend from l000mg/L in influent to about 260mg/L in effluent, and the removal ratio of scum is over 90%.
引文
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