The bacterial growth potential (BGP) of drinking water is widely assessed either by flow cytometric intact cell count (BGP_ICC) or adenosine triphosphate (BGP_ATP) based methods. Combining BGP_ICC and BGP_ATP measurements has been previously applied for various types of drinking water having high to low growth potential. However, this has not been applied for water with ultra-low nutrient content, such as remineralised RO permeate. To conduct a sound comparison, conventionally treated drinking water was included in this study, which was also used as an inoculum source. BGP_ICC, BGP_ATP, intact cell-yield (Y_ICC), and ATP-yield (Y_ATP) were determined for conventionally treated drinking water (Tap-water) and remineralised RO permeate (RO-water). In addition, both BGP_ICC and BGP_ATP methods were used to identify the growth-limiting nutrient in each water type. The results showed that the BGP_ICC ratio between Tap-water/RO-water was ∼7.5, whereas the BGP_ATP ratio was only ∼4.5. Moreover, the Y_ICC ratio between Tap-water/RO-water was ∼2 (9.8 ± 0.6 × 10⁶ vs. 4.6 ± 0.8 × 10⁶ cells/µg-C), whereas the Y_ATP ratio was ∼1 (0.39 ± 0.12 vs. 0.42 ± 0.06 ng ATP/µg-C), resulting in a consistently higher ATP per cell in RO-water than that of Tap-water. Both BGP_ICC and BGP_ATP methods revealed that carbon was the growth-limiting nutrient in the two types of water. However, with the addition of extra carbon, phosphate limitation was detected only with the BGP_ICC method, whereas BGP_ATP was not affected, suggesting that a combination of carbon and phosphate is essential for biomass synthesis, whereas carbon is probably utilised for cellular activities other than cell synthesis when phosphate is limited. It was estimated that the intact cell-yield growing on phosphate would be 0.70 ± 0.05 × 10⁹ cells/µg PO₄-P.

Measuring bacterial growth potential (BGP) involves sample pre-treatment and inoculation, both of which may introduce contaminants in ultra-low nutrient water (e.g., remineralized RO permeate). Pasteurization pre-treatment may lead to denaturing of nutrients, and membrane filtration may leach/remove nutrients into/from water samples. Inoculating remineralized RO permeate samples with natural bacteria from conventional drinking water leads to undesired nutrient addition, which could be avoided by using the remineralized RO permeate itself as inoculum. Therefore, this study examined the effect of pasteurization and membrane filtration on the BGP of remineralized RO permeate. In addition, the possibility of using bacteria from remineralized RO permeate as inoculum was investigated by evaluating their ability to utilize organic carbon that is readily available (acetate, glucose) or complex (laminarin, gelatin, and natural dissolved organic carbon), as compared with bacteria from conventional drinking water. The results showed that membrane filtration pre-treatment increased (140–320%) the BGP of remineralized RO permeate despite the extensive soaking and flushing of filters (>350 h), whereas no effect was observed on the BGP of conventional drinking water owing to its high nutrient content. Pasteurization pre-treatment had insignificant effects on the BGP of both water types. Remineralized RO permeate bacteria showed limitations in utilizing complex organic carbon compared with bacteria from conventional drinking water. In conclusion, the BGP bioassay for ultra-low nutrient water (e.g., remineralized RO permeate) should consider pasteurization pre-treatment. However, an inoculum comprising bacteria from remineralized RO permeate is not recommended as the bacterial consortium was shown to be limited in terms of the compounds they could utilize for growth.

Although water produced by reverse osmosis (RO) filtration has low bacterial growth potential (BGP), post-treatment of RO permeate, which is necessary prior to distribution and human consumption, needs to be examined because of the potential re-introduction of nutrients/contaminants. In this study, drinking water produced from anaerobic groundwater by RO and post-treatment (ion exchange, calcite contactors, and aeration) was compared with that produced by conventional treatment comprising (dry) sand filtration, pellet softening, rapid sand filtration, activated carbon filtration, and UV disinfection. The multi-parametric assessment of biological stability included bacterial quantification, nutrient concentration and composition as well as bacterial community composition and diversity. Results showed that RO permeate remineralised in the laboratory has an extremely low BGP (50 ± 12 × 10³ ICC/mL), which increased to 130 ± 10 × 10³ ICC/mL after site post-treatment. Despite the negative impact of post-treatment, the BGP of the finished RO-treated water was >75% lower than that of conventionally treated water. Organic carbon limited bacterial growth in both RO-treated and conventionally treated waters. The increased BGP in RO-treated water was caused by the re-introduction of nutrients during post-treatment. Similarly, OTUs introduced during post-treatment, assigned to the phyla of Proteobacteria and Bacteroidetes (75–85%), were not present in the source groundwater. Conversely, conventionally treated water shared some OTUs with the source groundwater. It is clear that RO-based treatment achieved an extremely low BGP, which can be further improved by optimising post-treatment, such as using high purity calcite. The multi-parametric approach adopted in this study can offer insights into growth characteristics including limiting nutrients (why) and dominating genera growing (who), which is essential to manage microbiological water quality in water treatment and distribution systems.

Ensuring the biological stability of drinking water is essential for modern drinking water supply. To understand and manage the biological stability, it is critical that the bacterial growth in drinking water can be measured. Nowadays, advance treatment technologies, such as reverse osmosis (RO), are increasingly applied in drinking water purification where the produced water is characterized by low levels of nutrients and cell counts. The challenge is, therefore, how to measure the low bacterial growth potential (BGP) of such ultra-pure water using the available methods which were originally developed for conventionally treated drinking water. In this study, we proposed a protocol to assess BGP of ultra-pure drinking water produced by RO and post-treatment (including remineralization). Natural bacterial consortium from conventional drinking water was added to all water samples during this study to ensure the presence of a wide range of bacterial strains. The method development included developing an ultra-pure blank with high reproducibility to lower the detection limit of the BGP method (50 ± 20 × 10³ intact cells/mL) compared with conventional blanks such as bottled spring water, deep groundwater treated by aeration and slow sand filtrate of surface water supply. The ultra-low blank consists of RO permeate after adjusting its pH and essential mineral content under controlled laboratory conditions to ensure carbon limitation. Regarding the test protocol, inoculum concentrations of >10 × 10³ intact cells/mL may have a significant contribution to the measured low levels of BGP. Pasteurization of water samples before measuring BGP is necessary to ensure reliable bacterial growth curves. The optimized method was used to assess BGP of ultra-pure drinking water produced by RO membranes and post-treatment (including remineralization), where the BGP has decreased more than 6-fold to a level of 90 ± 20 × 10³ intact cells/mL compared with conventionally treated water (630 ± 70 × 10³ intact cells/mL).