Elsevier

Water Research

Volume 142, 1 October 2018, Pages 227-235
Water Research

A uniform bacterial growth potential assay for different water types

https://doi.org/10.1016/j.watres.2018.06.010Get rights and content

Highlights

  • Bacterial community growth potential assay tested using six different sample types.

  • Indigenous communities from the sample were used as inocula.

  • Two suitable methods for assessing growth: FCM cell count and ATP analysis.

  • Reproducible results in triplicate growth tests.

  • Proportionality in growth with increase in carbon concentration for all sample types.

Abstract

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement.

This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed.

Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample.

The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon.

Introduction

Bacterial growth potential is the quantification of the extent of bacterial growth that can occur in a sample under defined conditions. Nutrient availability, mainly organic carbon and other growth-promoting/limiting compounds (e.g., nitrogen, phosphorus and iron), govern bacterial growth potential (Prest et al., 2016a; Nescerecka et al., 2018). Numerous methods to determine the bacterial growth potential and growth promoting properties of water have been developed throughout the last three decades (Van der Kooij et al., 1982; Servais et al., 1989; Hu et al., 1999; Ross et al., 2013; Prest et al., 2016a). The first developed methods for bacterial growth potential determination focused on the biodegradable organic carbon. The assimilable organic carbon (AOC) notion, initially proposed by Van der Kooij et al. (1982), is used to describe the portion of dissolved organic carbon (DOC) that is rapidly used by microorganisms to grow. AOC is viewed as an important parameter to assess the biological stability of water and the microbial growth potential during treatment and distribution (Srinivasan and Harrington, 2007; Baghoth et al., 2009; Hammes et al., 2010a; Weinrich et al., 2010; Kim et al., 2017). Unlike chemical methods to determine and characterize total organic carbon (TOC) or DOC, AOC explicitly targets a wide range of biologically available low molecular weight organic carbon compounds, generally present in low concentrations in water. The AOC bioassay is based on the linear relationship between the AOC concentration and maximum bacterial growth (i.e., maximum crop). For AOC calculations, a numerical yield factor (Y) is derived from the slope of a standard linear curve and is used to calculate the AOC concentration using the maximum bacterial growth of test bacteria. Determining the bacterial growth potential through the conventional AOC bioassay such as the assay by Van der Kooij et al. (1982) usually assumes organic carbon limitation which is not the case for all water samples. Several studies revealed that bacterial regrowth in drinking water in some regions was predominantly inhibited by inorganic phosphorous limitation (Sathasivan et al., 1997; Miettinen et al., 1999; Nescerecka et al., 2018). In these cases, determination of microbially available phosphorus (MAP), phosphorus that is readily assimilated by microorganisms, is more crucial than the AOC (Lehtola et al., 1999). In such waters, MAP is linearly correlated to bacterial growth potential, and a minor variation in the phosphorus concentration can have a major effect on the growth of bacteria. Therefore, growth potential bioassays were developed focusing on other possible microbial growth controlling substances which in some cases might be more crucial to describe and understand the bacterial growth potential rather than mainly organic carbon as the single growth-limiting substrate (States et al., 1985; Miettinen et al., 1997; Lehtola et al., 1999; Prest et al., 2016b; Nescerecka et al., 2018).

Numerous studies contributed to constantly optimize the bioassay with a main focus on three aspects: the selection of inoculum, the optimization of inoculation and incubation, and the evolution of bacterial growth measurements (LeChevallier et al., 1993; Sathasivan and Ohgaki, 1999; Wang et al., 2014; Van der Kooij et al., 2017). The conventional bioassays to measure the bacterial growth potential use selected pure cultures mainly Pseudomonas fluorescens P17 (P17) and Spirillum sp. NOX (NOX) as test strains primarily due to their abundance in water distribution systems and their ability to utilize organic carbon in low concentrations (Van der Kooij et al., 1982; Kaplan et al., 1993; LeChevallier et al., 1993). P17 and NOX require a simple nitrogen source and no growth-stimulating substances, such as vitamins. A major drawback of using pure cultures is the inability of some pure strains to universally grow in different water types (e.g., NOX does not grow in seawater) and to assimilate all the AOC present in the water. Moreover, the selection and use of specific single bacterial strains does not ensure similar results when different sample types are tested and changing the bacterial strains according to the sample type leads to results that are hard to compare. Therefore, the inoculum selection has been a point of focus in many studies and a principal alteration to the initial bacterial growth potential methods (Kemmy et al., 1989; Sathasivan and Ohgaki, 1999; Haddix et al., 2004; Weinrich et al., 2011); from using a single bacterial strain to a mix of two or more bacterial strains or the indigenous bacterial community. Some commercial assays and studies (Weinrich et al., 2011) used Vibrio harveyi bacteria instead of P17 and/or NOX to assess the growth potential of seawater which could not necessarily be used for freshwater. Indigenous bacteria demonstrated the ability to completely utilize the available AOC enabling a better estimate of the bacterial growth potential (Werner and Hambsch, 1986; Sathasivan and Ohgaki, 1999; Hammes and Egli, 2005; Prest et al., 2016a).

Shifting from cultivation dependent quantification methods to cultivation independent methods was another primary variation to the initial growth potential methods (LeChevallier et al., 1993; Hammes and Egli, 2005; Abushaban et al., 2017). Bacterial growth measurements evolved from the use of plate counting (Van der Kooij et al., 1982; Escobar and Randall, 2000) and turbidity measurements (Werner and Hambsch, 1986) to the use of adenosine tri-phosphate (ATP) luminescence method (LeChevallier et al., 1993; Van der Wielen and Van der Kooij, 2010; Van der Kooij et al., 2017), bioluminescence method (Weinrich et al., 2011), and total cell count with fluorescence staining and flow cytometry method (FCM) (Hammes and Egli, 2005; Gillespie et al., 2014; Wen et al., 2016). Flow cytometry (FCM) is a rapid bacterial cell counting tool for the assessment and evaluation of bacterial water quality. Adenosine tri-phosphate (ATP) dependent luminescence analysis is also viewed as a quick method for the measurement of viable microorganisms. The previously published FCM based AOC bioassay showed to be fast, reliable and reproducible (Hammes and Egli, 2005). With this approach, all bacteria in a water sample, including inactive or unculturable bacteria, can be quantified using total nucleic acid fluorescence staining of bacterial cells and FCM. The bioassay (Hammes and Egli, 2005) showed that with the application of an indigenous microbial community and incubation at 30 °C the stationary phase could be reached within 30–40 h following inoculation significantly reducing the time needed for AOC measurements.

This study aimed to provide an easy and uniform bacterial growth potential assay where different sample types are inoculated with their own bacterial community and growth is measured using less tedious and timesaving techniques: FCM and/or ATP. The suitability of the bioassay was investigated by evaluating the growth potential of six different microbial communities indigenous to the water being analyzed with increasing carbon concentrations. The effect of inoculating the same water with different indigenous microbial communities on the growth potential measurement was also examined. Measuring bacterial growth using total ATP luminescence was compared to FCM total cell counts for the different sample types for assessment of the suitability of the proposed methods under different sample types. A universal assay that is suitable to different sample type eases the implementation of the bacterial growth potential measurement, allows further understanding of bacterial growth dynamics in different sample types and facilitates comparison of results between different studies.

Section snippets

Preparation of carbon-free materials

Carbon-free bottles (Schott, Mainz, Germany) and vials (Supelco, Bellefonte, PA, USA) were prepared as described previously (Hammes and Egli, 2005). In short, all glassware was soaked overnight in 0.2 N HCl and subsequently rinsed properly with deionized water. After air-drying, the bottles and vials were baked in a Muffel furnace (500 °C; 6 h). Teflon-coated screw caps for the glassware were soaked overnight in acid (0.2 N HCl), thereafter in a 10% sodium persulfate solution (60 °C, 1 h),

Batch growth of an indigenous bacterial community

River water indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic matter without any carbon dosage. The batch growth of an indigenous river water bacterial community was determined using online FCM. The growth of the bacteria was relatively quick, with a lag phase of about 2 h and reaching the stationary phase in about 30 h (Fig. 2A). Exponential growth was recorded in all samples from as early as 5 h after inoculation. Fig. 2B displays

Growth potential test universal to all sample types

The uniform growth potential assay intends to provide a unified procedure that can be implemented for different sample types. The assay can measure the growth potential of different samples irrespective of the type of growth-limiting compounds. The use of indigenous bacterial community as inoculum is a focal point of the assay. Using the indigenous bacterial community present in the water tested as an inoculum demonstrated the ability to utilize a broader and diverse range of assimilable

Conclusions

The indigenous community bacterial growth potential assay showed consistent and reproducible bacterial growth results for all sample types tested providing clearer insights into the actual growth potential of different water types. The bioassay using FCM and ATP is fast, easy, and reliable therefore allowing the routine implementation of the growth potential measurement and providing a unified framework for different samples. The results showed:

  • Indigenous bacterial growth could detect low

Acknowledgements

The authors acknowledge the financial support of KAUST, Evides, and Eawag. The authors acknowledge Stefan Koetzsch, Joao Mimoso, and Hans-Ulrich Weilenmann for assistance with experiments and data analysis.

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