为了探讨生物质秸秆元素含量的快速检测方法，该文运用高光谱成像技术，结合多种数据优选方法对生物质秸秆中N、C、H、S、O元素含量快速检测的可行性进行研究。选取玉米、水稻、小麦、油菜4种类别共计188个秸秆样本，采集其反射高光谱图像，并测定元素含量。采用竞争性自适应重加权采样（competitive adaptive reweighted sampling，CARS）算法选取元素检测敏感变量，提取光谱维数据，结合偏最小二乘（partial least squares，PLS）算法，建立了基于高光谱光谱维数据的元素定量分析模型，N元素采用24个变量建立模型，验证集相关系数为0.923，均方根误差（root mean square error of validation set，RMSEP）为0.196%，相对分析误差（relative analysis error，RPD）为3.11；O元素仅采用10个光谱变量建立模型，验证集相关系数为0.876，均方根误差为1.015%，相对分析误差为2.32，N、O元素的模型可以用于实际应用；C、H、S元素相验证集相关系数均小于0.80，无法实际应用分析。采用独立成分分析（independent component analysis，ICA）算法结合权重系数法，提取IC1-IC5分析图像中特征光谱波段为572.09、643.69、685.14、766.79、819.55、964.01 nm，用6个特征光谱变量建立基于高光谱图像维数据的秸秆元素定量分析模型，N、C、H、S和O 5种元素无法用于实际检测。研究结果表明，采用高光谱成像技术并应用光谱维数据结合CARS-PLS算法可以实现秸秆N、O元素的有效检测。

Taking Trihoderma viride L4C as production strain， the influences of carbon source， nitrogen source， inoculum level， incubation time， cultivation temperature and initial pH value on cellulase activity produced by liquid fermentation were studied in this paper. On the basis of single factor tests， orthogonal test was made to study the optimal liquid fermentation conditions. The results showed that the incubation time had the largest effect on the yield of cellulase. The optimal condition was listed as follows: carbon source was the mixture of straw powder and cellulose powder， nitrogen source was ammonium sulfate， inoculum level was 15%， incubation time was 5 days， cultivation temperature was 28℃， initial pH value was 50. Under this condition， the activities of CMCase and FPAase were 1 31516 U·mL-1 and 1 28277 U·mL-1， respectively.

选取绿色木霉化L4C作为纤维素酶的生产菌株，分别研究了碳源、氮源、接种量、培养时间、培养温度和培养基初始pH对液态发酵方法产纤维素酶的影响。并在单因素试验的基础上，采用正交试验研究了绿色木霉化L4C液态发酵产纤维素酶的最佳培养条件。结果表明，培养时间对酶产量影响最大，液态发酵的最佳条件为：分别以稻草粉—纤维素粉混合物和硫酸铵为碳源和氮源，初始pH 50，接种量15%，28℃培养5 d。在最佳产酶条件下，羧甲基纤维素酶活性为1 31516 U·mL-1，滤纸酶活性为1 28277 U·mL-1。

[Objective] This study is aimed to obtain effective cellulose-degrading bacterial strains and study the characteristics of cellulase production and degradation characteristics used NH3?H2O pretreated corn stalk as substrate, and explore mechanism of cellulose enzyme so as to improve the resource utilization rate of agricultural solid wastes. [Methods] LB medium was used to obtain eleven bacterial strains (NH1?11) from earthworm farm. CMC-Na was used in preliminary medium and congo red staining method to screening strains. Influence of pretreatment to cellulose production ability of NH11 and degradation rate of substrates was studied. Morphological characteristics of NH11 was observed by electron microscope and identified by 16S rRNA and Biolog method. [Results] Bacterial strain NH11 was isolated and identified as Bacillus subtilis. The maximum degradation rate of untreated and pretreated corn stalk was 14.24% and 24.73% when culture temperature was 30 °C after five days. CMC cel-lulose activity of NH11 reached to 153.84 U/mL and FPA cellulose activity to 197.24 U/mL in treatment group, 11.45% and 10.59% higher than untreated group. [Conclusion] NH11 has a high cellulase productivity, and NH3?H2O pretreatment could enhance the degradation rate of corn stalk. NH11 has a high value in straw compost, mushroom culture medium and ruminant feed production.

【目的】获得高产纤维素酶细菌菌株, 探讨以氨化预处理玉米秸秆为底物时的纤维素酶产酶特性及底物降解特性, 探讨纤维素酶作用机理, 提高玉米秸秆利用率。【方法】用LB培养基分离并纯化菌株, 羧甲基纤维素钠培养基培养、刚果红染色进行初步筛选。考察氨化预处理对底物降解率、产酶能力的影响。通过形态特征观察及16S rRNA、Biolog鉴定菌株。【结果】分离到一株高效纤维素降解菌NH11, 经鉴定为枯草芽孢杆菌(Bacillus subtilis)。30 °C、发酵5 d时, 预处理前后玉米秸秆降解率分别为14.24%和24.73%。30 °C、pH 7.2时, 处理组CMC酶活力峰值处为153.84 U/mL, FPA酶活力为197.24 U/mL, 比未处理组分别高出11.45%和10.59%。【结论】NH11具有较高的纤维素酶产酶能力, 氨化预处理能够提高菌株对玉米秸秆的降解率。该菌株在秸秆堆肥、制作食用菌培养基和制取反刍动物粗饲料方面具有很高的应用价值。

Abstract: The decomposition of corn stalks returning to soil is very difficult. It has been the hot topic as to how to accelerate the decomposition of corn stalks in soil around the world. Isolation of cellulose-decomposing bacteria or fungi was conducted using the method of cellulose-Congo red stain in soil with straw returning which was collected from wheat-corn rotation farmland in Hebei province. The ITS genetic sequence of the strain determined by PCR was of 99% homology with that of Penicillium oxalicum when compared with the known sequence in the NCBI database using pairwise BLAST. The isolated strain was identified as Penicillium oxalicum on the basis of its morphological characteristics and ITS genetic sequence analysis and preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preserved number of CGMCC NO.4842. The ability to decompose corn stalks and the conditions to produce enzymes by this strain were studied further. The results showed that the enzymatic activities of CMC and FPA were 672.8, 282.9 and 774.6, 618.3U when the strain was growing in the sodium carboxymethyl cellulose (CMC-Na) medium and corn stalks in the medium at 30℃ for 72h, respectively. The total activity of cellulose on a corn stalks medium reached to 376.1U. Moreover, the optimum conditions for enzyme production were 3% beef extract peptone as the nitrogen source, 5% of inoculated level , 28-35℃, pH =4-7 and cultured for 48-96 h. The best combination of pH value, temperature, solid-liquid ratio, and incubation time was pH value=6.5, 30℃, solid-liquid ratio at 1:10, and incubation for 48h. When the strain was cultured with powder of corn stalks in a growth medium for 10 days, 87.3% of the corn stalks were decomposed and 1.90 times higher than that in the control. When the strain was incubated in soil with corn stalks for 30 days, the decomposition rate was 83.5% and 1.62 times higher than that in the control. Finally, the wheat pot experiment was carried out to investigate the effect of this strain on corn stalks decomposition in soil when the plant was grown for 50 days. The result showed that the straw decomposition rate was 70.8% and 15.1% higher than that in the control. In conclusion, the strain of Penicillium oxalicum isolated from the soil with straw returning could decompose the corn stalks efficiently.

目前还田的玉米秸秆存在腐解难的特点，如何加速玉米秸秆腐解成为国内外研究的热点。该试验采用纤维素-刚果红染色法从河北山前平原小麦-玉米轮作且长期秸秆还田的土壤中分离筛选到一株纤维素酶活性高的真菌。将测得的ITS基因序列与NCBI数据库上进行同源性比对，综合形态特征和ITS基因序列同源性分析，该菌株鉴定为草酸青霉（Penicillium oxalicum）。该文对其产酶条件和玉米秸秆分解能力进行了进一步研究。结果表明，在羧甲基纤维素钠（CMC-Na）液体培养基和玉米秸秆液体培养基上30℃培养72 h，内切葡聚糖酶（CMCase酶）和滤纸酶（FPase酶）活性分别达672.8、282.9和774.6、618.3 U；以玉米秸秆为底物其产天然纤维素总酶活性可达到376.1 U，说明该菌具有较强的玉米秸秆分解能力。该菌最佳产酶条件为：0.3%的牛肉膏蛋白胨为氮源，接种量为5%，培养温度为28～35℃，pH值=4～7，培养48～96 h；最优发酵条件为固液比为1∶10，培养时间为48 h，培养温度为30℃，pH值为6.5。该菌株对玉米秸秆腐解能力的研究表明，在秸秆揺瓶试验中，培养10 d后，秸秆腐解率达87.3%，为对照的1.90倍；在土壤培养试验中，培养45 d时，腐解率达83.5%，是对照的1.62倍；在玉米秸秆还田的小麦盆栽试验中，培养65 d时，秸秆腐解率达70.8%，高于对照15.1%。因此，从原位秸秆还田土壤中筛选出来的草酸青霉对玉米有较强的腐解能力。

为研究一种高效的玉米秸秆降解复合菌，选取了木质素降解优势土著菌密黏褶菌、环状芽孢杆菌、铜绿假单胞菌、栗褐链霉菌、黄孢原毛平革菌、杂色云芝、绿色木霉、黑曲霉，对各单一菌种对玉米秸秆的降解能力进行了测定，通过菌种间的拮抗试验，将单一菌种进行组合，初步构建了一组木质纤维素降解复合菌。结果表明：在整个35 d的预处理周期中，黑曲霉、绿色木霉对秸秆中纤维素、半纤维素体现了较高的降解能力，黑曲霉、绿色木霉对半纤维素的降解率分别为47.81%、37.53%，对纤维素的降解率分别为38.96%、46.32%；黄孢原毛平革菌、杂色云芝对玉米秸秆中的木质素体现了较强的降解能力，对木质素的降解率分别为43.56%、39.17%；菌种拮抗试验表明该试验所选用的真菌、放线菌及细菌之间无拮抗反应，可以进行混合培养；对复合菌预处理前后的玉米秸秆微观结构进行扫描电镜分析，发现在降解过程中复合菌对木质纤维素的结构产生了破坏作用，提高了木质纤维素的可及性；木质素、纤维素、半纤维素的含量在整个发酵过程中都在逐渐减少，发酵结束时复合菌对半纤维素的降解率最高达到48.53%，纤维素的降解率为36.38%，木质素的降解率为40.11%，在提高木质素降解率的同时减少了纤维素消耗。该研究为秸秆类生物质降解及利用提供了参考依据。

Twelve strains were isolated from putrid straw,wood,soil and cattle feces etc. They can grow in a solid medium with(sodium carboxymethyl cellulose) CMC-Na as an only carbon source.One strain of fungi,preliminarily identified as Neurospora sp.,with a strong ability to decompose cellulose was screened from the 12 strains by a series of experiments.The experiment of single factor optimization about the solid-state fermentation of this strain was carried out.The research results shows that to the strain,the optimal carbon and nitrogen sources are that the mass ratio of straw to bran is(9 to 1,) and NH 4 NO 3 0.5%;and that the optimal conditions for culture are that the inoculum’s size is 1 mL,the ratio of solid to liquid 1 to 3,culture temperature around 30 ℃ and cultivating time 96 h under the condition of a natural pH value.The carboxyl methyl cellulase activity and the filter paper activity of Neurospora sp.are 3551.1 and 386.7 μmol/(g·h) respectively under these optimal zymotic conditions.

从腐烂稻草、土壤和牛粪等样品中分离到12株能在以CMC-Na(羧甲基纤维素钠)为唯一碳源的固体培养基上生长的菌株,并从中筛选出1株分解纤维素能力较强的真菌,初步鉴定为脉纹孢菌( Neurospora sp.).通过对其固态发酵条件进行单因素优化试验,初步确定了发酵培养基最佳碳、氮源种类及添加量:稻草粉与麸皮的质量比为9;1,NH 4 NO 3 0.5%;最适培养条件为:固液比1;3,接种量1 mL,pH值自然,培养温度30℃,培养时间96 h.该菌株的羧甲基纤维素酶活和滤纸酶活分别达到3 551.1和386.7μmol/(g.h).