Abstract: 9217 A. Introduction

1. Significance

The growth of bacteria in drinking water distribution and storage systems can lead to the deterioration of water quality, violation of water quality standards, and increased operating costs. Growth or regrowth in these systems results from viable bacteria surviving the disinfection process and metabolizing nutrients in the water and biofilm to sustain growth.¹ Factors other than nutrients that influence regrowth include temperature,² residence time in mains and storage units,³ and the efficacy of disinfection.⁴ Tests to determine the potential for bacterial regrowth focus on the concentration of nutrients.^5–7

Not all organic compounds are equally susceptible to microbial decomposition. The biodegradable organic matter (BOM) fraction that provides energy and carbon for bacterial growth has been called labile dissolved organic carbon,^8,9 biodegradable dissolved organic carbon (BDOC),⁷ or assimilable organic carbon (AOC).⁵ Easily measured chemical surrogates for BOM are not available.^10,11 Chemical methods alone are inadequate for BOM estimation because of the tremendous diversity of natural organic matter, involving thousands of potentially degradable molecules^12,13 and the abilities of microorganisms to utilize nutrients and energy sources present a very low concentrations.¹⁴ As alternatives to chemical methods, bioassays have been proposed^15-17 in which heterotrophic bacteria, the dominant organisms responsible for the degradation of BOM, are used as the inoculum. Some bioassay methods focus on measurements of the growth of the microbial inoculum, while others measure the activity of the microbes by assessing chemical changes in the test water. In a bioassay based on growth, the growth of a bacterial inoculum to maximum density can be used to estimate the concentrations of limiting nutrients. The underlying assumptions of the AOC bioassay are that nitrogen and phosphorus are present in excess, i.e., that organic carbon is limiting, and that the bioassay organisms represent the physiological capabilities of the distribution system microflora. Various bioassay procedures use an inoculum of 1 to 4 species of bacteria^{5,15,16,18-20} growing in log phase or present in late stationary phase, or may use undefined bacteria attached to a sand substratum,⁷ suspended in the sample,^6,21 or filtered from the sample and then resuspended.¹⁷ Incubation vessels vary as to material,²² size,^23,24 closure,²³ and cleaning procedure.^5,23,24 Water to be tested for nutrient concentrations has been variously prepared.^5,7,17 The AOC bioassay is an indirect or surrogate method, wherein nutrient concentrations are not measured directly, but through colony-forming units (CFUs) of the bioassay organisms. Nutrient concentrations have been estimated directly from changes in dissolved organic carbon concentrations in the test vessel⁷ or indirectly from epifluorescence microscopic counts of the maximum number of bacterial cells grown,^16,17 turbidity,¹⁷ or incorporation of tritiated thymidine into bacterial DNA.^6,25 Since the first publication of the AOC method, additional approaches have been defined including total cell counts,²¹ ATP content,²⁶ or bioluminescence.²⁰ CFU densities, total cell densities, or bacterial production are converted to nutrient concentration by the growth yield of bacteria, defined as either the ratio between CFU or cells produced and organic carbon used, or biomass produced and organic carbon used.^5,6