Effect of different heterotrophic plate count methods on the composition of culturable microbial community

Heterotrophic plate counts (HPC) are routinely determined within the scope of water quality<br>assessment. However, variable HPC methods with different cultivation conditions are applied.<br>These variabilities can have a significant effect on the outcome of the analysis, particularly with<br>regard to the composition of culturable microbial community. This was demonstrated by testing<br>commonly used low and high incubation temperatures (22 and 37°C) as well as high and low<br>nutrient media (yeast extract agar and R2A). The culturable microbial community composition was<br>investigated by 16S rRNA gene sequencing and additional statistical analysis (ANOVA permutation<br>test on CCA and PERMANOVA). Lower temperature(22°C) showed the abundance of naturally<br>occurring Pseudomonadaceae and Aeromonadaceae, whereas at high temperature (37°C)<br>numerous Enterobacteriaceae and Bacilli were identified. The highest biodiversity was detected at<br>lower temperature, especially on R2A medium. The most significant effect of tested cultivation<br>conditions was assigned to temperature (p &lt; 0.01), although media also affected the abundance of<br>certain detected bacteria (as seen in the number of operational taxonomic units). These results<br>endorse the supposition that different temperatures (low and high) should be included into HPC<br>measurement, whereas the selection of media should take into account the monitored water<br>source itself. Accordingly it can be inferred that the HPC method is more suitable for continuous<br>monitoring of water quality than for single assessments.

异养平板计数（Heterotrophic plate counts, HPC）是水质评估领域的常规检测项目。然而当前应用的培养条件各异的平板计数方法存在诸多差异，这类差异会对分析结果产生显著影响，尤其会影响可培养微生物群落的组成结构。
本研究通过测试常用的高低培养温度（22℃与37℃）以及高低营养培养基（酵母提取物琼脂（yeast extract agar）与R2A培养基）验证了上述结论。研究采用16S rRNA基因测序及附加统计学分析（CCA置换方差分析（ANOVA permutation test on CCA）与置换多元方差分析（PERMANOVA））对可培养微生物群落组成进行了探究。
较低温度（22℃）条件下，自然存在的假单胞菌科（Pseudomonadaceae）和气单胞菌科（Aeromonadaceae）丰度更高；而在高温（37℃）条件下，则可检出大量肠杆菌科（Enterobacteriaceae）与芽孢杆菌纲（Bacilli）物种。
在较低温度下，尤其是使用R2A培养基时，检测到的生物多样性最高。
在所测试的培养条件中，温度对实验结果的影响最为显著（p < 0.01），尽管培养基也会对部分检出细菌的丰度产生影响（可从可操作分类单元（operational taxonomic units, OTU）的数量变化中体现）。
上述结果佐证了一项推论：异养平板计数检测应纳入高低不同的温度条件，而培养基的选择则需结合待测水源的自身特性。据此可推断，相较于单次水质评估，异养平板计数方法更适用于水质的连续监测。