Microbiological changes along a modular wastewater reuse treatment process with a special focus on bacterial regrowth

Water reuse is becoming an increasing necessity due to depleted water resources or increased water demand. A treatment process on a pilot scale was designed to produce different water qualities for different applications in industry or agriculture. We report heremicrobiological changes along themodular process using treated municipal wastewater effluent as raw water. Treatment technologies included coagulation, ultrafiltration (UF), reverse osmosis (RO), quartz sand, activated granular activated carbon (GAC) filtration and disinfection. Elimination of traditional hygiene indicator bacteria was already achieved by ultrafiltration as the first barrier. Profound changes by each treatment step also applied to the microbiome. Total and intact cell concentrations as quantified by flow cytometry underwent a strong decline after UF and RO, whereas biological stabilization was achieved through quartz sand filtration and GAC passage. Interestingly assimilable organic carbon (AOC) was still present even after RO at levels that allowed substantial regrowth of bacteria. Overall, UF and RO led only to a 0.43 and 0.78 log decrease in intact cells concentrations in stagnated water after regrowth compared with 6.5 log intact cells/ml in the stagnated rawwater. Temperaturewas shown to be an important parameter determining themicrobiome of the regrown population. Regrowth could, however, be efficiently suppressed by monochloramine.

cytometry) was only reduced moderately (on a log scale) despite a strong reduction of DOC (on a linear scale).
• Flow cytometry proved a very sensitive diagnostic tool both in an offline and online format.

INTRODUCTION
Climate change leads to prolonged dry weather periods and extending semi-arid regions and poses an increasing stress for cities, industries and agriculture in many regions worldwide (Ormerod ; Faour-Klingbeil & Todd ). Also, in Germany that has previously not been associated with water shortage, local water limitations have become more obvious in the recent dry summers. To avoid excessive stress for groundwater sources, water reuse is increasingly debated especially for industrial uses and irrigation (Drewes & Horstmeyer ; Schmid & Bogner ; Drewes et al. ). Given the different types of water uses and the different corresponding water quality requirements, water treatment concepts should ideally be flexible and adjustable to needs (Becker et al. ). Further challenges consist in the allocation of variable demand-driven water volumes at competitive prices.
To gain experience with how to accomplish different quality requirements in such a scenario, a project named 'MULTI-ReUse' was launched. A coastal region with competing water uses (demand from water-intensive industries versus demand for drinking water) was chosen for a demonstration. As groundwater use would come with the risk of saltwater intrusion due to its direct proximity to the North Sea coast, this region in the northwest of Germany does not have its own drinking water supply. Instead, drinking water is transported over long distances. Whereas relatively low water qualities would be sufficient for most industrial applications and agriculture, high-quality drinking water is used in many cases. Water reuse would, thus, be an immediate alleviation on drinking water demand, groundwater resources elsewhere and energy transport costs. Aims of the project consisted of the (1) optimization and validation of modular treatment trains to achieve different water qualities, (2) improvement of process monitoring strategies, (3) assessment of economic and ecological aspects and socio-cultural acceptance and (4) development of marketable treatment solutions for globally relevant and typical reuse applications.
For this purpose, a German multicenter research consortium developed, demonstrated and evaluated a combination of different water treatment technologies to polish treated obtained within the time stagnation time (Gillespie et al. ). The bacterial levels obtained after the 7 days are supposed to correlate with the assimilable organic carbon (AOC) and nutrient levels contained in the sample (Farhat et al. ). Indirectly the approach, therefore, also allows conclusions on the efficiency of nutrient removal.
In summary, this study aimed at monitoring changes in the numbers of bacteria along a modular water reuse treatment process and the composition of the bacterial population. The analysis was not limited to the microbiological status of the water directly after sampling, but also tried to cover the changes in that water after stagnation. Two UF modules and two RO lines, each with three modules and recycling of concentrate, were operated in parallel to test different process conditions during an initial test (1) UF filtrate, (2) RO permeate and (3)  decanted (for recycling purposes) and glassware and caps were rinsed three times with tap water and twice with deionized water. Dried glassware and caps were wrapped in aluminum foil. Glassware was muffled at 280 C for at least 8 h or overnight, caps were heated at 180 C during that time.

Sampling procedure
Water samples were taken from designated sampling valves after disinfection with isopropanol (70%) followed by desiccation for at least 2 min (following guidelines by DIN EN Offline flow cytometry Fluorescent dyes used in this study were SYBR Green I (10,000× stock, Invitrogen™) and propidium iodide (PI, 1 mg/ml, Invitrogen, Thermo Fischer). Water samples were processed undiluted or 10× diluted with 0.1 μm filtered mineral water (Evian, Evian-les-Bains, France) in case the total signal exceeded approximately 5.000 signals/s. SYBR Green I was diluted to a working stock concentration of 100× using dimethylsulfoxide (DMSO, Sigma-Aldrich) and stored at À20 C until use. Aliquots (250 μl) of water samples were transferred into 96-well plates (cat. nr. 601808, HJ-Bioanalytik GmbH). To determine total cell concentrations (TCC), 200 μl sample aliquots from this plate were transferred into the wells of a second 96-well plate with pre-aliquoted 2 μl of the 100× SG working stock solution followed by thorough mixing by pipetting up and down several times using a multichannel pipette. To determine ICC, 200 μl sample aliquots from the first plate were transferred into the wells of a second 96-well plate with pre-aliquoted 2.4 μl mixture of a 100× SG I and PI in the ratio of 5:1 again followed by thorough mixing. Final concentrations of fluorescent dyes were 1× SG and 3 μM PI.
Staining was performed at 37 C for 13 min in an incubator.
Data were collected using an ACEA NovoCyte ® benchtop instrument equipped with a 488 nm laser (OLS OMNI Life Science, Bremen, Germany). A NovoSampler ® Pro enabled analysis on a 96-well plate basis. Data were analyzed using the instrument-specific NovoExpress software and a gating procedure similar to the one described by Gatza et al. ().

Online flow cytometry
Online flow cytometry was performed using an Online Bacteria Analyzer (OBA, ONTRONIX AG, Switzerland). A stain solution with a total volume of 100 ml was prepared by mixing 2 ml PI (1 mg/ml), 0.1 ml SYBR Green I (10,000×), 10 ml DMSO and 87.9 ml ultrapure water to give concen- Phyloseq in combination with ggplot (version 3.2.1) and Inkscape (version 0.92) (http://www.inkscape.org/) were used for plotting and illustration.

Assessment of the hygienic status
The most important criterion of water is its hygienic safety. Traditional bacterial hygiene indicators and bacterial colony counts as quantified by culture methods were abundant in treated wastewater effluent serving as raw water for the pilot plant (Table 1)    the filtrate side. The bacterial community underwent further change within the UF filtrate tank and on its way to RO, consistent with a moderate increase in cell numbers (Figure 2).
In case of the sand filter and GAC passage of UF filtrate, diversity and species richness (407 and 600 OTUs after sand filter and GAC, respectively) increased significantly in agreement with biological stabilization. Overall, the bacterial microbiome was shown to be highly dynamic and to undergo various substantial changes along the treatment train reflected in a Bray-Curtis dissimilarity plot (Figure 3  value of raw water on that day. This example illustrates that traditional AOC assessment has a detection limit. Whereas the parameter is useful at measurable concentrations, AOC values near zero on a linear scale can still translate to a substantial number of bacteria on a log scale. Even under extremely oligotrophic conditions bacteria can reach concentrations in the range of 10 5 -10 6 cells/ml (Egli ). It has to be acknowledged in this context that the traditional AOC assay is optimized for potable water. To improve the assay's suitability for reuse applications, the two bacteria Pseudomonas fluorescens P17 and Spirillum sp. NOX forming the basis of the drinking