Nelson Cassandra E, Beri Nina R, Gardner Jeffrey G
Department of Biological Sciences, University of Maryland - Baltimore County, Baltimore, MD, USA.
Department of Biological Sciences, University of Maryland - Baltimore County, Baltimore, MD, USA.
J Microbiol Methods. 2016 Nov;130:136-143. doi: 10.1016/j.mimet.2016.09.013. Epub 2016 Sep 21.
Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.
对顽固多糖降解进行生理学研究具有挑战性，原因有几个，其中之一是使用不溶性底物获得可重复准确的细菌生长实时测量存在困难。当前方法存在几个问题，包括：（i）由于不溶性物质与细胞交织而导致背景噪声高；（ii）耗材和试剂成本高；（iii）采样与数据采集之间存在显著时间延迟。一种可定制的底物和细胞分离装置将为利用光密度测量研究细菌生长提供一种选择。为了验证这一假设，我们使用3D打印制造了生物质容纳装置，该装置允许不溶性底物与微生物细胞相互作用，但不干扰分光光度计测量。对可用于3D打印的材料进行评估表明，紫外线固化丙烯酸塑料是最佳材料，在耐热性、反应性和可灭菌能力方面优于尼龙或不锈钢。对3D打印装置的成本分析表明，与活细胞计数或蛋白质测量相比，它们是定量细菌生长的一种有竞争力的方法，并且实验条件可在100倍范围内扩展。使用可溶性底物或不溶性底物时，该装置的存在不会改变生长表型。我们应用生物质容纳来表征日本纤维弧菌在真实木质纤维素（未预处理的玉米秸秆）上的生长，并发现生理学证据表明，尽管存在大量纤维素，但木聚糖是一种重要的营养来源。
