CN113302477A - 使固体表面上的生物膜可视化和量化的方法 - Google Patents

使固体表面上的生物膜可视化和量化的方法 Download PDF

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CN113302477A
CN113302477A CN202080008425.0A CN202080008425A CN113302477A CN 113302477 A CN113302477 A CN 113302477A CN 202080008425 A CN202080008425 A CN 202080008425A CN 113302477 A CN113302477 A CN 113302477A
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G·马森
G·吉尔艾伯特奥里奥尔
V·加西亚莫利纳
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Abstract

一种用于使固体表面上的生物膜可视化的方法。所述方法包括以下步骤：(a)提供固体表面，所述固体表面一直与水性介质接触并且所述固体表面的至少部分可能地覆盖有生物膜，(b)将所述固体表面保持在水平位置并用碳颗粒水性分散体覆盖所述固体表面，(c)使所述固体表面以与水平呈至少5的角度倾斜，以允许过量的碳颗粒水性分散体从所述表面流下，(d)通过确定是否有未被碳颗粒水性分散体覆盖的区域来检测所述固体表面上存在的任何生物膜。

Description

使固体表面上的生物膜可视化和量化的方法
背景技术
本发明总体上涉及一种用于使固体表面、尤其是聚合物表面上的生物膜可视化和量化的方法。
反渗透(RO)和纳滤(NF)膜原件中的生物膜生长(生物积淤)仍然还是RO/NF市场中的关键挑战之一。细菌产生的生物膜阻塞可能造成跨过RO模块的压降升高，从而导致液压失衡并且可能使模块受损。此外，生物膜可能影响膜传递特性并且产生跨膜压力(TMP)降低，这降低了通量。这些影响中的每一个不但增加了操作能量，而且还会导致频繁清洗(CIP)以恢复膜性能。典型的方法涉及用染料使生物膜着色，这可能是不可逆的并且还有害于膜。例如N.Sreedhar等人在Desalination[脱盐]，2018，425：12-21中报告了用结晶紫给膜着色的方法。本发明旨在提供一种用于增强生物膜结构的可视化和量化、不会对生物膜结构或膜产生永久性影响的手段。
发明内容
本发明涉及一种用于使固体表面上的生物膜可视化的方法。所述方法包括以下步骤：
(a)提供固体表面，所述固体表面一直与水性介质接触并且所述固体表面的至少部分可能覆盖有生物膜，
(b)将所述固体表面保持在水平位置并用碳颗粒水性分散体覆盖所述固体表面，
(c)使所述固体表面以与水平呈至少5°的角度倾斜，以允许过量的碳颗粒水性分散体从所述表面流下，
(d)通过确定是否有未被碳颗粒水性分散体覆盖的区域来检测所述固体表面上存在的任何生物膜。
具体实施方式
除非另有说明，否则所有百分比是重量百分比(wt％)，并且所有温度以℃计。除非另有说明，否则平均值是算术平均值。在室温(18℃至25℃)下进行实例中的所有操作，除非另有说明。优选地，本发明的方法在3℃至45℃、优选地10℃至35℃的温度范围内进行。
优选地，碳颗粒水性分散体为“墨汁”，其是以此名称销售的作为碳颗粒水性分散体的产品。优选地，碳颗粒具有不大于2微米；优选地不大于1微米、优选地不大于0.7微米的算术平均直径。优选地，颗粒具有至少0.01微米、优选地至少0.05微米；优选地至少0.1微米的算术平均直径。上限和下限是可组合的。优选地，水性分散体的碳含量为0.5至10wt％、优选地1至9wt％、优选地2至8wt％。水性分散体可以含有少量其他物质，例如粘合剂、表面活性剂等。
优选地，固体表面是聚合物表面，优选地膜，例如反渗透、纳滤或超滤膜。优选地，形成聚合物表面的聚合物是聚酰胺(例如包含间苯二胺或哌嗪和均苯三甲酰氯的聚合单元)、聚酯(例如聚对苯二甲酸乙二醇酯)或烯烃；优选地是聚酰胺。
优选地，将水性分散体施用到处于水平位置的固体表面，并且然后将表面倾斜以除去多余的墨。优选地，将少量墨放置在处于水平位置的聚合物表面的角上。优选地，添加到表面的墨的量为基于该表面的面积至少0.1mL/cm2、优选地至少0.11mL/cm2、优选地至少0.12mL/cm2、优选地至少0.13mL/cm2。墨的最大量不是决定性的，因为比所需的多的量无非是在表面倾斜时从表面流下来。典型地，所需的量不多于0.2mL/cm2。优选地，将表面倾斜，以便在使用时使墨水沿液体流过该表面的方向流动。优选地，表面以与水平呈至少20°、优选地至少30°；优选地不超过80°、优选地不超过60°、优选地不超过50°、优选地不超过40°的角度倾斜。优选地，表面倾斜的时间为2至40秒、优选地至少5秒、优选地至少10秒；优选地不超过30秒、优选地不超过20秒。优选地，具有生物膜的表面的面积是通过目测观察、优选地借助于数字摄影和数字图像处理而计算确定的。
优选地，用于表面生物膜量化的图像处理涉及以下步骤：
(a)处理数字图像，优选地借助于图像处理软件
(b)将图像转换成8位灰度级，优选地通过将颜色通道拆分成RGB色彩空间，然后优选地选择绿色层并将其转换成8位灰度级
(c)将图像转换为1位，优选地通过将黑色阈值从255调节到当像素数的一阶导数/颜色范围等于或低于0(简单移动平均值为10个周期)时的颜色范围。
(d)通过用1位空间中的生物膜像素(白色像素)除以整个像素数(PT)来计算生物膜表面覆盖百分比
优选地，膜是从下方照亮的以增强存在生物淤积的区域(其呈现为清晰且明亮的点)的对比和可视化。优选地，待测聚合物表面具有至少100勒克斯、优选地至少300勒克斯；优选地不超过4000勒克斯、优选地不超过3000勒克斯的平均亮度。
实例
对于着色样品的宏观目视检查，使用莱卡MS5立体显微镜(0.63x至4x的放大倍率)来允许样品的真实可视化。该仪器使用带有两个物镜和目镜的两条独立光路。这会形成稍微不同的视角，以产生样品的三维可视化。还能够通过在物体下方放置灯泡进行透射光照明。使用照明是因为其增强了存在生物淤积的区域的对比和可视化。用相机捕获的样品的面积从7.6至510.7mm2不等，取决于所使用的立体显微镜中的放大倍率水平。使用用于图像采集的12兆像素数码相机获得的照片的分辨率估计为10μm左右，这与莱卡MS5立体显微镜的衍射极限一致。该技术是基于在1位图像上，将对应于生物膜(白色)的像素与背景(黑色)的像素分离开。
(a)用于验证此方法的生物淤积样品自经受生物淤积的RO试样获得。将来自RO试样的4cm×4cm湿润样品水平放置在陪替氏培养皿中。使用双面带将膜样品与陪替氏培养皿固定在一起。将2mL的PELIKAN Black Fount India制图墨(百利金公司(Pelikan)，瑞士)(算术平均颗粒尺寸：0.4微米)放置在一直与过滤的原水接触的膜的一侧。
(b)倒入墨后，将样品以与水平呈30°倾析10秒钟以除去多余的墨
(c)借助立体显微镜(莱卡MS5)使生物淤积的样品可视化。所使用的立体显微镜的放大倍率为2x，光强度为500勒克斯
(d)使用12.1兆像素数码相机(佳能数码Ixus 200 IS)获得数字照片并使用ImageJTM 1.51软件处理照片
(e)将颜色拆分成RGB通道。然后选择绿色层并将其转换成8位灰度级
(f)通过将黑色阈值调整为11O使图像转换成1位照片，这会产生能够使生物膜覆盖率可视化的图像。该图像在12,000,000个像素中含有2,151,502个白色像素，这表示生物膜表面覆盖率为18％。

Claims (8)

1.一种用于使固体表面上的生物膜可视化的方法；所述方法包括以下步骤：
(a)提供固体表面，所述固体表面一直与水性介质接触并且所述固体表面的至少部分可能覆盖有生物膜，
(b)将所述固体表面保持在水平位置并用碳颗粒水性分散体覆盖所述固体表面，
(c)使所述固体表面以与水平呈至少5°的角度倾斜，以允许过量的碳颗粒水性分散体从所述表面流下，
(d)通过确定是否有未被碳颗粒水性分散体覆盖的区域来检测所述固体表面上存在的任何生物膜。
2.如权利要求1所述的方法，其中，所述固体表面是聚合物表面。
3.如权利要求2所述的方法，其中，所述碳颗粒水性分散体是墨汁。
4.如权利要求3所述的方法，其中，所述生物膜通过获得包含碳颗粒水性分散体的生物膜的摄影图像而检测。
5.如权利要求4所述的方法，其中，对所述摄影图像进行数字处理以产生1位的图像。
6.如权利要求5所述的方法，其中，使所述固体表面以与水平呈至少20°的角度倾斜。
7.如权利要求6所述的方法，其中，将所述碳颗粒水性分散体以至少0.1mL/cm2的量添加到所述表面上。
8.如权利要求7所述的方法，其中，所述碳颗粒水性分散体具有0.5至10wt％的碳含量。
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