Name: isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture
Uploaded: 2013-05-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

斯蒂芬·阿尔德曼博士，博士大卫叶和Erin马克里表示诚挚的感谢与显微镜的帮助。通过赠款资助这项研究是从的NIFA特色作物研究计划，USDA得离谱SREP格兰特奖2009-02484号。梅根·莱昂哈德约瑟夫·麦科勒姆，凯文·金洛克，BRIANA Kinash玛丽亚McSharry和妮可做人提供了优良的技术援助和周到的讨论。

此过程需要至少几个月孵化BDM片土壤中，还有几个个月的时间顺序琼脂平板上和琼脂，肉汤评估已有化学定义殖民化和降解的生物测定。个人的方法列出的顺序，他们将被执行。 1。 BDM薄膜在土壤中孵化纳入BDM片土壤条件下，模仿下，他们将有望降低。收购400克（干重当量）居民的土壤和三明治一个10厘米×10厘米的BDM一个13×13厘米的尼龙网眼袋（250微米）...

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这里所描述的步骤，表示第一通潜在BDM从土壤中降解菌的分离技术，并已成功用于分离真菌从埋在土壤中为7个月的BDMS。真菌生长时，再接种到新鲜的的BDM材料的相同类型的，表明分离的真菌确实殖民者，薄膜不抑制真菌生长。塑料降解真菌和细菌的分离可能导致它们的使用，单独或组合使用，修订土壤或堆肥塑料需要被降解。 在他们的原生土壤环境中，微生物物种中不存在...

历来被认为是降解塑料聚合物的不良属性，，因为击穿缩短产品的寿命和耐久性。近日，废塑料在自然环境1,2,3环境问题的意识已经由生物可降解塑料有吸引力的替代传统的塑料材料。退化（定义为结构的变化，碎裂，和分子量降低，完整性，和强度4,5）通过一系列的事件，包括非生物过程（热应力，光氧化，水解，侵蚀和机械应力）发生，和生物降解。虽然非生物过程可以改变片段的大小和特性的塑料，微生物所需的最终矿化水和二氧化碳（在有氧条件下）和/或甲烷（在厌氧条件下）。 有相当利基生物降解性塑料存在于农业，塑料覆盖物的使用，以防止杂草生长，保持土壤水分，增加土壤温度7,8。仅在美国，数千英亩的数百覆盖有塑料覆盖物，包括可生物降解的塑料覆盖物组成。在作物生长季节，出售的可生物降解的覆盖物（BDMS）的选项包括出售在一个垃圾填埋场，焚烧能量回收10，通过堆肥降解或降解在土壤耕作后11。其中，至少劳动力密集型的命运春耕BDMS到土壤中，但没有有效的降解和矿化在非作物个月期间（一般在冬季），塑料碎片可能保持和干扰农业设备在春季耕作和种植，坚持环境，让他们显着影响的野生动物，植物的生命和微生物1,2,3,10。 12。在冬季，大多数地区的土壤温度将低于这些温度，可能导致甚至更低的微生物的活动，因此，矿化。除了 ​​减缓退化率，误用术语“生物降解”，导致这些产品的不信任，消费者13,14，包括那些在农业产业。生物降解转换二氧化碳（和/或甲烷）和水14由自然发生的微生物4的聚合物。因此，生物降解必须的化学计量;微生物与基材的物理缔合，并不意味着该材料的微生物降解。 审查可持续利用农业BDMS的努力的一部分，这个研究主要集中在发现原生微生物农业定植和降低市售BDMS的的的的土壤。已经出版了化学测量破裂的生物降解塑料，生物和非生物的手段15,16,17的标准试验方法。然而，这些方法都没有解决个别微生物物种的降解塑料，或它们的隔离方法。本文的方法更接近于标准方法旨在真菌孢子接种标本后评估抗微生物击穿塑料18,19。 当塑料的配方是已知的，原料的来源提供的，粉状塑料，可以暂停由可视化结算区13在以琼脂为基础的媒体和退化。该方法已被用于先前识别微生物降解的聚（丁二酸丁二醇酯- 共 -己二酸酯）的聚合物，如聚氨基甲酸酯20，21，和聚（乳酸）22。类似的方法，包括纯塑料粉末悬浮在液体培养基中，塑料是唯一碳源20,23。虽然这些方法有一个定义的系统的优点是，他们不良模仿BDMS 原位降解。首先，BDMS，因为没有分散在整个土壤基质中的小颗粒，而是，出售，用作薄膜的表面积不同的分布。其次，不同于纯聚合物的化学组成BDMS的。 BDMS通常含有添加剂，如填料，增塑剂，着色剂，这些添加剂可能会影响微生物的生长，从而，矿化率。出于这个原因，因为在这项研究中的某些商业片的组成是专有的，在现场准备的塑料薄膜，是用来隔离真菌和细菌。为简单起见，在下面的方法，仅描述为真菌，经过修改的注意到在适当的情况下对细菌隔离。 在最近的一项研究24，三市售BDMS的一个实验电影在美国的三个不同区域的农业网站，一个生长季节，随后放置在目（250微米）袋和埋在土壤中的一个冬天在同一站点。 250微米筛孔允许真菌菌丝穿透，同时不包括根和大多数土壤动物，并尽量减少土壤侵蚀25,26。尼龙材料，防止塑料袋在​​土壤中的降解。经过挖掘，Fungal菌株中回收由BDM件和没有碳的来源除了琼脂和5厘米×5厘米表面的灭菌新的未使用的的BDM膜，预灭虫平方评估在基本培养基上生长。大多数塑料用作电影不能高压灭菌不丧失诚信，所以UV光被用来杀死任何塑料上的微生物细胞。 ISO 846 19建议在70％乙醇和随后的干燥表面灭虫，但如果使用此方法，必须确保没有的薄膜的组分或添加剂带来不利影响，乙醇。由于BDMS大概制造承受阳光，紫外线被选为去污方法。 BDM片优于仅在基本培养基上生长的菌株作进一步研究。琼脂，海藻产生的多糖，是用来凝固微生物的介质，因为它通常是利用代谢农业和医学上不能力的微生物，但是，琼脂水解酶已被分离自海洋细菌27和28从土壤中也已被隔离的水解琼脂细菌。有BDM聚合物与琼脂均是罕见的土壤真菌，包含这些聚合物作为潜在的营养源的环境中，还没有发展所分泌的酶的底物，但本文所述的（ 步骤7）中的板生物测定，存在两个基板。使用的BDMS，但不琼脂作为碳源的真菌可以区别于真菌只使用琼脂，通过比较生长在琼脂固体培养基含有琼脂培养基（阴性对照）， 二除外）没有添加碳源）BDM片（实验）以及iii）葡萄糖（阳性对照）。预期生长的影响所有菌株在基本培养基上附加葡萄糖真菌所产生的葡萄糖的板可能有其他的实验中所用的特定的基本培养基中生长的能力。电位人BDM降解菌生长在琼脂固化的基本培养基+ BDM电影更好，比他们单独琼脂凝固的基本培养基上生长。培养基平板上生长的真菌是琼脂降解菌或oligotrophs的，预计也将生长在琼脂与BDM电影在生物测定板，而不是在自己的电影（除非他们偶然也会降低BDM聚合物）。 为了消除可能看到到利用琼脂和不BDMS的由于微生物的生长，我们遵循我们的初步检测BDM定植琼脂平板上定义的肉汤培养基（ 第9步 ）生物测定。 BDM枚较的生物测定管中已知的唯一的碳源。 经过初步筛选，并呼吁恢复甘油股票的菌株，有些形成很少，但可见菌丝体液体介质中含有任何已知的碳源。这些结果表明，一些所获取的菌株oligotrophs -生物体生长的清除非常少量的碳，氮，和其他营养物质溶解在含水环境中的或现有的在空气中的挥发物29,30,31。物种鉴定通过18S核糖体DNA分析支持这一观点，许多相匹配的菌株真菌属先前报道表现出贫营养32。 ，这是常见的腐生oligotrophs，范围广泛的环境中30的范围内的底物利用的代谢能力的要求。因此，这并不奇怪，我们孤立从BDMS（大概需要不寻常的酶的能力）相同的真菌表现出贫营养的能力，并能够成长的痕量污染物如皮肤沾染指纹，灰尘，或在空气中的微量挥发油。由于隔离oligotrophs，我们得出的结论是不能单独用来推断BDM击穿增长一个BDM表面上。本文所述的方法反映我们的努力，SCREEN本地的BDM殖民者从农业土壤善意 BDM击穿。

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

原产于的农业土壤殖民市售的生物降解地膜（BDM）薄膜的真菌的分离和降解塑料的潜力进行评估。通常，当塑料的配方是已知的，原料的来源提供的，粉末状的塑料可以悬浮在以琼脂为基础的媒体和退化，由可视化的信息交换区。然而，这种方法不佳模仿BDMS 原位降解。首先，BDMS不分散作为整个土壤基质的小颗粒。其次，BDMS商业销售作为纯的聚合物，而是作为含有添加剂（ 如填料，增塑剂，染料），可能会影响微生物的生长的薄膜。本文所述方法，用为收购土埋地膜的菌株。真菌菌株从挖掘BDMS获得被单独测试的增长奠定了新的，灭虫BDMS的定义中不含碳源电子顶上件琼脂XCEPT。菌株进一步测试增长BDMS的液体培养基中，BDMS唯一碳源。约10周后，真菌和BDM退化评估通过扫描电子显微镜。通过核糖体RNA基因序列的分析，分离和鉴定。本报告介绍了真菌隔离的方法，但细菌也适合细菌代媒体使用这些方法进行分离。我们的方法应该是有用的研究完整的塑料薄膜或产品，塑料原料是未知或不可用故障。然而我们的方法并不提供定量的方法比较BDM降解率。

在最近的一项研究24个重复每三个市售BDMS的标有“可降解”，再加上一部实验电影和传统塑料控制，放在土壤覆盖番茄生产在2010年的春天在华盛顿州Mount Vernon，诺克斯维尔，田纳西州，德克萨斯州拉伯克。在2010年的秋天，BDM电影广场被切断每个风化覆盖4个重复的情节，乡土辗转直接从下方的区域覆盖物样品已切除。每个风化BDM方形夹在土壤内的尼龙网袋，如步骤1所述。只?...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

塑料薄膜标有“可降解”市售作为覆盖物用于农业用途。耕作是一个有吸引力的处置方法，但在野外条件下的降解是知之甚少。这项研究的目的是开发用于隔离本地土壤真菌和细菌后场埋葬殖民塑料地膜的方法。

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

科研

生物学

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

可重复的阶梯步骤测定，以访问杂草水稻的过敏性潜力（Oryza sativa ssp.）

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...