Manganese removal processes during start-up of inoculated and non-inoculated drinking water biofilters

Manganese removal in drinking water bio ﬁ lters is facilitated by biological and physico-chemical processes, but knowledge regarding the relative role of these mechanisms during start-up is very limited. The aim of this study was to identify the dominant process for manganese removal occurring during the start-up period of sand ﬁ lters with and without inoculation by addition of matured sand collected from an operating groundwater-based waterworks. Inoculation with matured ﬁ lter sand is frequently used to accelerate the start-up in virgin bio ﬁ lters and to rapidly obtain compliant water quality. The non-inoculated ﬁ lter took 41 days to comply with manganese quality criteria, whereas the inoculated ﬁ lter with 20% matured sand showed removal from Day 1 and compliance from Day 25. By Day 48, the inoculated ﬁ lter showed two times higher manganese removal rates and manganese oxides deposits. Using sodium azide as an inhibitor of microbial activity, it was found that manganese removal in the non-inoculated ﬁ lter was dominated by biological processes, whereas physico-chemical processes were of more importance in the inoculated ﬁ lter (Day 35, 39 and 48). 16S rDNA sequencing of the microbiota collected during ﬁ lter maturation indicated a limited immediate effect of inoculation on the microbial community developed on the remaining ﬁ lter material.


INTRODUCTION
Biofilters are often used in production of drinking water from groundwater sources. However, a major disadvantage of biofiltration is the necessity of a start-up period to mature virgin filter media. When manganese is present in source water, the start-up period can last from weeks to more than a year (Tekerlekopoulou et al. ).
Proactive inoculation methods to accelerate the start-up of biofilters include the addition of a concentrated source of microorganisms and/or autocatalytic surfaces, e.g. backwash sludge (Štembal et  The aim of this study was to identify the dominant process in manganese removal (physico-chemical and biological) during the start-up of a virgin sand pilot biofilter with and without inoculation by addition of matured sand.
In addition, the aim of this study was to shed light on the effect of proactive inoculation by addition of a layer of matured filter sand on the microbial community developed in the adjacent virgin layers of the filter.

Pilot scale set-up
Treated groundwater from the storage tank of a Danish drinking water treatment plant was used as source water (Fredensborg waterworks, Skanderborg, Denmark). The treated water contains a natural background of drinking water microorganisms as disinfection is not used at this and most other waterworks in Denmark. The unchlorinated treated water was continuously spiked with a concentrated solution of MnCl 2 · 4H 2 O (Emsure ACS), using a diaphragm pump (Digital DDC, Grundfos), and distributed to two pressurized 0.3 m 3 filter tanks (Type NS20, Silhorko Eurowater). The filters were placed at the water treatment plant and operated at a temperature of 11 C (Table 1, Figure 1).
Each filter has a diameter of 30 cm and a 1 m layer of granular quartz sand. The non-inoculated filter was filled solely with virgin quartz sand (Dansk Kvarts Industri), and the inoculated filter with two intercalated layers of matured sand in the virgin sand ( Figure 1, Table 2). The matured sand used for inoculation of the pilot filter was collected from the top layer of a second stage biofilter of Fredensborg waterworks, which had been removing manganese for the last 47 years. Throughout the experiment, no visual mixing was observed between the two-filter media used in the inoculated filter (virgin and matured sand).
Before the start of operation, the filter tanks and the virgin sand medium were disinfected overnight with 2% H 2 O 2 according to the manufacturer's standard procedures   Water and filter medium sampling Inlet and outlet samples (10 mL) from the filters were manually collected each couple of days for a period of 72 days, filtered (0.22 μm) and analyzed immediately for total dissolved manganese. In addition, water samples were collected from both filters at 10 cm depth intervals (profile) once a week following the same procedure. Inlet water (4 L) was filtered (0.20 μm membrane filters, Advantec) and stored at À21 C for subsequent microbial diversity analysis.
Before the start of operations (Day 0) virgin sand and matured sand samples were collected to quantify the manganese coating the grains, to investigate the manganese removal rate and to analyze the microbial diversity. Photometer, Thermo Fisher Scientific). All bottles were continually mixed on an orbital shaker (150 rpm) at room temperature during the experiment. Control bottles without filter medium, with and without NaN 3, were included to account for any precipitation or sorption of manganese to glass surfaces of the incubation bottles.
Column assay with and without NaN 3 for determining manganese removal rates The non-inoculated filter (Figure 3(a)) initially showed no detectable manganese removal (Day 6 and 20) followed Manganese removal at the upper third of the inoculated filter was greatly increased by Day 35 (Figure 3(b)). Interestingly, the amount of manganese removed from the 10-20 cm layer in the inoculated filter (initially virgin sand) was similar to the amount removed from the 0-10 cm layer (initially mature sand). This suggests that manganese removal capacity was transferred from the mature sand to the virgin sand layer directly underneath.
It should be noted that removed manganese at 0-10 and 10-20 cm layers diverged near the end of the experiment, not because the 0-10 cm layer was more efficient, but due to the 10-20 cm layer receiving lower manganese concentrations. Downloaded from https://iwaponline.com/wqrj/article-pdf/54/1/47/669491/wqrjc0540047.pdf by guest that comparable amounts of manganese were removed by those layers (Figure 3(b)). These results suggest that accumulation of approximately 1% (≈0.1 mg/g) of the manganese coating the matured filter sand was sufficient for the initially virgin sand to achieve a performance comparable to the fully matured sand. These results indicate that freshly precipitated manganese oxide on the initially virgin filter is more efficient than precipitates on the initially matured grains.

Manganese coating in inoculated and non-inoculated
At Day 48, the amount of manganese coating on the initially virgin medium at 20 cm and 30 cm depth of the inoculated filter was twice as high as the non-inoculated filter ( Figure 4). The same two-fold difference was observed when comparing the total manganese removed by the 10-20 cm layer of each filter by Day 48 (20 cm depth, Figure 3(c)).

Manganese removal with and without NaN 3 in batch and column assays
NaN 3 is an inhibitor of respiratory activity in microorganisms while it does not appear to affect autocatalytic properties of MnOx coatings (Rosson et al. ). NaN 3 addition was used in the current study to compare manganese removal processes related to physico-chemical and biological mechanisms. Manganese removal observed in medium samples with NaN 3 was assumed to be mainly due to physico-chemical processes, and the difference between the manganese removal observed in medium samples with and without NaN 3 was assumed to be mainly due to biological processes ( Figure 5). To identify the dominant process in manganese removal, the ratio between apparent physico-chemical and biological removal rates was calculated. When the ratio was <1, most manganese removal was attributed to biological processes, and when the ratio was >1 most manganese removal was attributed to physico-chemical processes.
Medium samples collected from the non-inoculated filter during the start-up period of manganese removal indicated that the manganese removal was attained by both biological and physico-chemical processes (Day 35 and 39, Figure 5(a)).
During that period, the ratio between physico-chemical and biological removal rates was 1 on average and showed no statistical difference over depth (p > 0.05 after Mann-Whitney test). In contrast, physico-chemical removal mechanisms appeared to dominate after the start-up period at the deeper biofilter layers (depth 20 and 30 cm) with an average ratio of 15, whereas biological mechanisms remained important for manganese removal at 10 cm depth of the filter with an average ratio of 0.5 (Day 48, Figure 5(a)).
The manganese removal rate of the non-inoculated filter at Day 48 due to physico-chemical processes was three times higher at 20 cm than at 10 cm depth (Figure 5(a)). Similarly, the amount of manganese coating on the grains of the noninoculated filter at Day 48 was 3.5 times higher at 20 cm than at 10 cm depth (0.162 mg Mn/g medium and 0.046 mg Mn/g medium respectively, Figure 4).
In contrast to the non-inoculated filter, no time dependent change was observed in the manganese removal processes occurring in the inoculated filter. Filter medium samples from all depths showed that the manganese removal from Day 35 to Day 48 was mostly due to physico-chemical processes ( Figure 5(b)). Further, the ratio between physico-chemical and biological removal rates showed no statistical difference over depth (p > 0.05 after Mann-Whitney test).

The initially matured sand layers in the inoculated
filter more than doubled their manganese removal rates over time from Day 35 to Day 48. This increase in manganese removal at 10 cm depth (initially matured sand) of the inoculated filter was also observed in the manganese profiles (Figure 3(b)). From Day 39, the initially virgin sand layer at 20 cm depth of the inoculated filter showed similar manganese removal rates as media samples from the initially matured sand layers (0-10 cm and 50-60 cm, Figure 5(b)). This similar performance in manganese removal at 10 cm and 20 cm depth (initially matured sand and initially virgin sand, respectively) was also observed in the manganese profiles of the inoculated filter (Figure 3(b)).
Overall, the sum of the physico-chemical and biological manganese removal rates in media samples from the inoculated filter was on average two times higher than the total manganese removal rates from the non-inoculated filter ( Figure 5(a) and 5(b)). A two-fold difference between the filters was also observed at 20 cm depth by Day 48 in the total manganese removal of the pilot filters (Figure 3(c)) and in the MnOx coating (Figure 4). The ratio between physico-chemical and biological removal rates of all medium samples was statistically different between the filters (p < 1 × 10 À11 after Mann-Whitney test), with a 0.8 median for the non-inoculated filter indicating a more active role of biological processes, and a median of 4.3 for the inoculated filter suggesting a more active role of physico-chemical processes ( Figure 5(c)).
A column assay with and without NaN 3 addition was Hence, the results obtained in the current study complement previous investigations of matured filters, suggesting that the physico-chemical processes in manganese removal increase with filter medium age. • From the onset of manganese removal to compliance, both physico-chemical and biological processes contributed to the manganese removal in the non-inoculated filter. One week after compliance, biological mechanisms remained important for manganese removal at the top 10 cm of the non-inoculated filter, whereas physicochemical processes were of more importance at deeper filter layers.

Bacterial diversity in inoculated and non-inoculated
• The major manganese removal processes occurring in the filters were statistically different. The non-inoculated filter was dominated by biological processes, whereas physico-chemical processes were of more importance in the inoculated filter. Inoculation appeared to mainly enhance the physico-chemical manganese removal potential during start-up.
• The use of proactive inoculation by addition of matured filter sand contributes to a shorter start-up period of biofilters with limited effect on the microbial community developed in the adjacent layers of the filter.