Biocarriers Improve Bioaugmentation Efficiency of a Rapid Sand Filter

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Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide (BAM)-contaminated drinking water Benjamin Horemans, Bart Raes, Johanna Vandermaesen, Yanti Simanjuntak, Hannelore Brocatus, Jeroen T'Syen, Julie Degryse, Jos Boonen, Janneke Wittebol, Ales Lapanje, Sebastian R. Sorensen, and Dirk Springael Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05027 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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Environmental Science & Technology

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Title: Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide (BAM)-contaminated drinking water

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Running title: Biocarriers for remediating BAM-contaminated drinking water

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Authors: Benjamin Horemans1,*, Bart Raes1, Johanna Vandermaesen1, Yanti Simanjuntak1, Hannelore Brocatus1, Jeroen T’Syen1, Julie Degryse2, Jos Boonen2, Janneke Wittebol3, Ales Lapanje4,5, Sebastian R. Sørensen6,7, Dirk Springael1

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1

Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KULeuven, Kasteelpark Arenberg 20 bus 2459, 3001 Heverlee, Belgium

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De Watergroep, Vooruitgangstraat 189, 1030 Brussel, Belgium

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Bioclear, Rozenburglaan 13, 9727 DL Groningen, The Netherlands

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Josef Stefan Institute, Jamova 49, Ljubljana, Slovenia

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National Research Saratov State University, Astrakhanskaya 83, Saratov, Russian Federation

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Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, 1350 Copenhagen K, Denmark

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Bacterial Discovery, Novozymes A/S, Krogshoejvej 36 DK-2880 Bagsvaerd, Denmark (present address)

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* Corresponding author: [email protected]

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Dr. Ir. Benjamin Horemans

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Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee,

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Belgium.

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Phone: +32(0)16329675; fax: +32(0)16321997

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e-mail: [email protected]

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Keywords: 2,6-dichlorobenzamide, bioaugmentation, immobilization, Aminobacter sp. MSH1, micropollutant

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1 ACS Paragon Plus Environment


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Abstract

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Aminobacter sp. MSH1 immobilized in an alginate matrix in porous stones, was tested in a

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pilot system as an alternative inoculation strategy to the use of free suspended cells for

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biological removal of micropollutant concentrations of BAM in drinking water treatment

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plants (DWTPs). BAM removal rates and MSH1 cell numbers were recorded during operation

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and assessed with specific BAM-degradation rates obtained in lab conditions using either

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freshly grown cells or starved cells to explain reactor performance. Both reactors inoculated

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with either suspended or immobilized cells showed immediate BAM removal under the

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threshold of 0.1 µg/L but the duration of sufficient BAM removal was two-fold (44 days)

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longer for immobilized cells. The longer sufficient BAM removal in case of immobilized cells

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compared to suspended cells was mainly explained by a lower initial loss of MSH1 cells at

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operational start due to volume replacement and shear. Overall loss of activity in the

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reactors though was due to starvation and final removal rates did not differ between

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reactors inoculated with immobilized and suspended cells. Management of Assimilable

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Organic Carbon in addition to cell immobilization appears crucial for guaranteeing long term

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BAM degradation activity of MSH1 in DWTP units.

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Introduction

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Pesticides are widespread micropollutants in freshwater environments and pose a challenge

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for drinking water production plants (DWTPs)1, 2. European countries largely depend on

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groundwater for drinking water provision, while stringent threshold pesticide residue

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concentrations in drinking water of 0.1 µg/L for individual compounds have been

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implemented in the European Union (EU)3. As such, pesticide residue concentrations in

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groundwater above the EU threshold limit has important implications for drinking water

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production such as the costly closure of abstraction wells or the inclusion of expensive

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treatment measures such as activated carbon (AC) filtration or oxidation with NaClO or H2O2

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in DWTPs4. More economic and sustainable approaches to remove pesticide residues in

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DWTPs are needed1. Remediation relying on microbial biodegradation processes in DWTP

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biofiltration units is perceived as a viable alternative involving either indigenous or added

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pesticide degrading organisms2. Bioremediation of micropollutant contaminated intake

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waters in DWTP filtration units is challenging though due to the intrinsic low carbon and


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nutrient content of groundwater5, shear stress due to high flow rates, grazing by protozoa6, 7

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and competition of the degraders with indigenous sand filter organisms for space and

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nutrients8.

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2,6-dichlorobenzamide (BAM) is a highly mobile and recalcitrant transformation product of

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the widely used herbicide dichlobenil (2,6-dichlorobenzonitrile)9-12. BAM is frequently found

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in groundwater at concentrations in the micropollutant range12-14 exceeding the EU drinking

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water limit. Abstraction wells are either closed or BAM is removed with activated carbon

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(AC) filtration units in DWTPs. Aminobacter sp. MSH1 is an aerobic soil isolate that uses BAM

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as sole source of carbon, nitrogen and energy14,

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microconcentrations as low as 1.5 µg/L14, 16. The conversion of BAM to 2,6-dichlorobenzoic

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acid (DCBA) is catalyzed by the amidase BbdA encoded by the bbdA gene17. The constitutive

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expression of bbdA assures theoretically that BbdA is continuously present in MSH1 cells

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while its high affinity for BAM17 implicates that MSH1 is an efficient catalyst for BAM-

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degradation at micropollutant concentrations.

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Strain MSH1 was proposed for use in bioaugmentation of treatment units employed in

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DWTPs treating BAM contaminated intake water. Emphasis was on the sand filter units

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implemented for removal of iron and manganese oxides in DWTPs18. Sand filter units are

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common use in DWTPs and provide a matrix that allows biofilm formation of a wide range of

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microbiota19. Slow sand filters treat the water at a flux of 0.4 m/h, while rapid sand filters do

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so at a flux of 4-8 m/h20. The inoculated MSH1 cells have to colonize the quartz grains of

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sand filters under oligotrophic conditions and high shear stress. BAM removal will occur as

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long the loss due to inefficient attachment, shear and cell death is compensated by growth

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of cells with sufficient activity. In the oligotrophic environment of DWTPs, heterotrophic

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bacteria will mainly depend on assimilable organic carbon (AOC) for growth and

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maintenance21 which is estimated in groundwater around 100 µg C/L5.

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A recent pilot study examined the use of MSH1 in rapid sand filters for treating water

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contaminated with BAM at trace concentrations (0.2 µg/L) in continuous conditions22.

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Bioaugmentation was performed by mixing a MSH1 cell suspension with sand of the top

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layer of the reactor22. Adequate BAM removal below the EU threshold limit of 0.1 µg/L was

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achieved but only in the first twenty days after inoculation. The decrease in BAM removal

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was suggested to be due to cell loss due to shear stress, grazing by protozoa and decay22, 23.

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and mineralizes the compound at



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Successful introduction of MSH1 in the sand filters might be improved by using alternative

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cell delivery approaches, e.g., using porous beads or gel carriers for immobilization which

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might reduce shear stress and/or grazing by protozoa24-26.

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In this study, pilot scale sand filter reactors were tested for treating BAM contaminated

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groundwater using Aminobacter sp. MSH1 cells immobilized in an alginate matrix on/in

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porous stones. BAM-removal was monitored and MSH1 cell numbers quantified by real time

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quantitative PCR (qPCR) during operation to estimate MSH1 cell loss from the systems.

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Results were compared to reactors amended with free suspended cells.

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Material and methods

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Strain and culture conditions. Aminobacter sp. strain MSH1 (CIP 110285)14 was stored at -

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80°C in glycerol (15% v/v). Cultures for bioaugmentation were started from colonies grown

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on R2A27 amended with 200 mg/L BAM (25°C, 5 days). BAM was added to assure BAM-

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catabolic stability of the MSH1 cultures.

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Site description and pilot scale sand filter setup. The pilot system was installed at a DWTP in

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Egenhoven, Belgium, where extracted groundwater is contaminated with 0.2 μg/L BAM. The

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pilot scale sand filter module included four PVC columns (see Supplementary Information

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(SI) Figure S1) with 12.3 cm diameter and 200 cm length. The columns were filled with a

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layer of 15 cm pebble stones (3-5 cm), covered by a polyamide filter and further filled with

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the appropriate filter bed material up to a 130 cm height (16500 cm³ total volume). On top

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of the filter bed, a water column of 20 cm was maintained. Groundwater collected in a

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storage tank was pumped by a Watson-Marlow 520S peristaltic pump to the top of the

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columns. All four columns were first filled with coarse sand (0.8-1.5 mm) as filter bed

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material with no MSH1 added and percolated with groundwater at a flow of 7.5 L h-1 for

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three months (November 2013 until January 2014) to evaluate BAM behaviour in the system

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and optimize system hydraulics. Two bioaugmentation experiments were performed. In both

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experiments, MSH1 was added either as suspended free cells or immobilized cells.

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The first bioaugmentation experiment (exp. 1) was performed from February to April 2014.

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The free cell suspension used for inoculation, was obtained by culturing strain MSH1 in 10 L

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of R2B containing 200 mg/L BAM while aerated using a magnetic stirrer (300 rpm) for 2 days

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at 25°C. The culture was harvested (6000 x g, 15 min.), washed with 10 mM MgSO4 and 4 ACS Paragon Plus Environment

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resuspended in 1 L of 10 mM MgSO4 at a cell density of 1013 cells/L. Coarse sand (0.8–1.5

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mm) was used as filter bed material and bioaugmentation was performed by stopping the

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flow and pouring the 1 L cell suspension at the top in the filter bed. After 1 hour the flow

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was resumed. The immobilized cell inoculum was obtained as follows. Strain MSH1 was

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grown at 20oC in mineral salt medium MSNCopt containing glucose for 5 days at 200 rpm28.

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The cells were washed three times in 0.9% NaCl by centrifugation and suspended in

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ultrapure water (MilliQ™) containing 0.5% Na-alginate at a cell density of 2 x 107 MSH1

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cells/mL. Porous stones (Matrix, Seachem, USA) were washed in ultrapure water (MilliQ™),

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autoclaved at 170oC for 1h, air dried for one day at 60°C and submerged in the MSH1-

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alginate suspension (0.5 L of cell-alginate suspension/kg stones). Three times a vacuum of

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70% of atmospheric pressure (30 kPa) was applied resulting in air release from the pores of

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the stones after which atmospheric pressure was restored to suck in the cell-alginate

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suspension in the pores (14%w/w/stone mass). The carriers were washed with ultrapure

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water (MilliQ™) and 0.2 M CaCl2 solution (0.5 L/kg of carriers) was added to form the Ca2+-

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alginate matrix entrapping the MSH1 cells in and on the stones. The MSH1 containing

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carriers were stored at 4°C until use (7 days in Exp 1). Bioaugmentation with the immobilized

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inoculum was performed as follows. Coarse sand was removed from the column and the

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carrier material containing the immobilized MSH1 cells was mixed with part of the removed

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coarse sand (0.8–1.5 mm) in a 44:56 volume ratio and added to the column after which flow

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was initiated. The two remaining columns acted as non-bioaugmented reactors, i.e., with

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column C1 containing coarse sand and column C2 containing a mixture of coarse sand and

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the porous stones used for MSH1-carrier production (in a 44:56 volume ratio) (Table 1, SI

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Figure S1). The total amount of added MSH1 cells to the reactors were for both strategies

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around 1013 with numbers verified in the inoculum prior to inoculation using bbdA targeted

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real time qPCR as described below. During operation, Hundred mL influent and effluent

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water samples for chemical analysis and total germ counts were taken weekly. Additionally,

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hundred mL influent and effluent samples were taken weekly for BAM concentration

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analysis and kept at 4°C in glass vials containing 100 mL of 0.5 vol% 37% HCl until analysis.

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Duplicate effluent samples (100 – 200 mL) for DNA extraction were collected in sterile

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bottles (Shott) from day 0. The samples were filtered over a WhatmanTM glass microfiber

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filter which was stored at -20°C. Duplicate filter bed samples (approx. 20 mL) for DNA

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extraction were taken weekly from the top of the column (first 20 cm). Samples of reactors 5 ACS Paragon Plus Environment


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amended with suspended MSH1 cells were taken from day 0 (1 h after start), samples from

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reactors containing immobilized cells were taken from day 4 (corresponding to 0.7 m³ of

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treated water). Two hundred fifty mg portions of the sampled sand material were stored at -

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20°C until DNA extraction. At the end of the experiment, sand filter samples were taken

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from the reactor inoculated with free suspended cells, layer by layer every 7-13 cm and 250

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mg duplicate samples of sand material were stored at -20°C. No final stage samples were

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stored/analyzed of the reactor containing immobilized cells because of reactor failure

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several days before harvest.

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The second experiment (Exp 2) was conducted from June to October 2014. The MSH1 free

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cell suspension was obtained by growing strain MSH1 first in 100 mL of MS medium29

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containing 200 mg/L BAM for 7 days. The culture was transferred to 10 L R2B amended with

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200 mg/L BAM and incubated with aeration using a magnetic stirrer (300 rpm) for 2 days at

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25°C. MSH1 was first precultured on medium containing only BAM to increase selection for

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MSH1 cells that can mineralize BAM. This suspension was used to inoculate R2B containing

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BAM for obtaining a high cell density culture. As in Exp. 1, coarse sand was used as filter bed

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material and the free cell suspension was applied to the top layer (Table 1). In contrast to

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Exp 1, the cell suspension was recirculated over the column for 5 days before operation was

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started. Bioaugmentation with immobilized cells was performed as described for Exp 1 using

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the same MSH1 carriers but 6 months after production. As in Exp 1, the remaining two

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columns were used as as non-bioaugmented control reactors (Table 1, SI Figure S1). As in

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Exp 1, the total number of added MSH1 cells to the reactors were for both strategies around

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1013 with numbers verified in the inoculum prior to inoculation using bbdA targeted real

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time qPCR as described below. Influent and effluent water samples for chemical analysis,

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total germ counts were taken as in Exp 1. Effluent water and top layer sand filter material

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was sampled and prepared for DNA extraction as in Exp 1. In addition, duplicate samples of

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carrier material were taken from reactors amended with immobilized cells from the top

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layer during operation. Carriers were separated from the sand, the carrier material was

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crushed using a DNA-free mortar and 250 mg portions were stored at -20°C for DNA

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extraction. At the end of the experiment, sand filter samples, and, in case of the reactor

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containing immobilized cells, also carrier samples, were taken layer by layer every 7-13 cm


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and 250 mg duplicate samples of sand material and/or crushed carrier material were stored

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at -20°C.

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Chemical analysis and total germ count. Non-purgeable organic carbon (NPOC) was

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determined according to procedure ISO 8245:1999. Nitrogen (ammonia, nitrite and nitrate)

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and ortho-phosphate concentrations were determined spectrophotometrically (ISO 10304-

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1:2007). Total germ counts (CFU/mL) were determined by plating on yeast extract agar

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medium and incubation at 22°C (48 hour incubation) and 37°C (72 hour incubation) (ISO

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6222:1999). BAM concentrations were determined by UPLC-MS/MS (LOD of 20 ng/L)

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following procedure WAC/IV/A/02730.

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DNA extraction and qPCR analysis. DNA extraction from effluent, sand and carrier samples

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was performed using the PowerSoil® DNA Isolation Kit (Mo Bio Laboratories, Inc, USA)

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following manufacturer guidelines. Extracted DNA eluted in 50 µL elution buffer, was stored

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at -20°C until qPCR analysis. MSH1 abundance was quantified (two technological replicates)

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by qPCR targeting the bbdA gene as reported in SI. bbdA encodes for BbdA that converts

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BAM to DCBA and represents a selective marker for MSH1 cells that perform BAM-

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degradation17. The plasmid pBAM1 is a low copy number plasmid (1 to max. 3/cell). Gene

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copy numbers are expressed per cm³ of column volume (suspended cell strategy: gene

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copies/g sand x 0.68 cm³/g sand x 0.44 volume sand/column; immobilized cell strategy:

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copies/g sand x 0.68 cm³/g sand x 0.28 volume sand/column for the sand fraction and

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copies/g carrier x 0.30 cm³/g carrier x 0.44 volume carrier/column for the carrier fraction).

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The LOQ of the bbdA qPCR was 105 copies/cm³ of column volume and 106 copies/liter

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effluent.

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Regression analysis and statistical analysis

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Significant differences (confidence interval 95%) for bbdA numbers (qPCR) (duplicate

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/reactor, two technological replicates) from different reactor configurations were

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determined with unpaired Student’s T-test and between different time points within the

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same reactor configuration with paired Student’s T-test. Logarithmic (total cells) and power

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(cells in effluent) regressions were performed with SigmaPlot 12.0 (Systat Software inc).

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Significance of estimated regression coefficients (95% confidence intervals) was determined



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with ANOVA. Total MSH1 cell numbers, cell loss, cell loss rates during the experiments, and

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groundwater AOC were calculated using the measured BAM removal rates as described in SI.

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Results

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Intake water characteristics. The chemistry of the groundwater extracted in Egenhoven-

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West showed little variation in time (SI Table S1). The C:N:P ratio was around

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144(±110):1592(±114):1(±0). At all sampling time points, BAM concentrations exceeded the

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drinking water threshold of 0.1 µg/L with an average concentration of 0.19±0.04 µg/L.

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Atrazine (0.02±0.01 µg/L; 37 out of 42 sampling occasions) and desethylatrazine

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(0.023±0.001 µg/L; 22 out of 42 sampling occasions) were occasionally detected.

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BAM removal efficiency and MSH1 dynamics in sand filter reactors either inoculated with

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free suspended MSH1 or immobilized MSH1 (Exp 1). A first experiment was performed to

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examine whether bioaugmentation with immobilized cells improves BAM removal

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performance compared to bioaugmentation with free suspended cells. We hypothesized

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that bioaugmentation with immobilized cells would increase the longevity of BAM

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degradation till below the threshold limit of 0.1 μg/L compared to bioaugmentation with

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free cells due to an improved retention of the inoculum in the system. Non-inoculated

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control reactors containing the same material but without added MSH1 cells were included

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to assure removal of BAM is due to MSH1 activity.

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In the reactor inoculated with free suspended MSH1 cells, BAM was detected in the effluent

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at concentrations under the EU threshold of 0.1 µg/L for at least the first 3 m³ of filtered

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water (17 days) (Figure 1). After the first 3 m³ of treated water, effluent BAM concentrations

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exceeded the threshold to reach gradually a BAM removal efficiency of around 20% upon

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treating 8 m³ (44 days). In the reactor inoculated with immobilized cells, BAM concentrations

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in the effluent were under the EU threshold of 0.1 µg/L for most of the time period and only

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reached the threshold at the end of the experiment, i.e., upon treating 8 m³ (44 days) water

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(Figure 1). No BAM removal occurred in the non-inoculated control reactors. At day 44, the

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experiment had to be aborted because of pump failure.

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During operation, bbdA numbers were enumerated in the top layer of the filter material (SI

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Figure S2) and the effluent (SI Figure S3). In the reactor inoculated with free cells, bbdA 8 ACS Paragon Plus Environment

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numbers were initially (at day 0) 3.6±0.4 x 109 bbdA/cm³ in the top layer and decreased

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linearly with a factor 100 to reach finally 3.1±0.4 x 107 bbdA/cm³ after 3 m³ of treated water.

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In the effluent, an exponential decrease in bbdA numbers occurred from 1.5±0.1 x 1011 to

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2.6±0.3 x 106 bbdA/L reaching stable numbers after the first 3 m³ of treated water. In case of

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immobilized cells, bbdA numbers were only determined in the sand fraction and not in the

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carriers. bbdA numbers in the sand fraction in the top layer remained constant at around

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4.2±1.7 x 106 and 3.4±5.4 x 106 bbdA /cm³ of sand. In the effluent, bbdA numbers decreased

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from 1.2±0.0 x 109 until 3.2±0.1 x 107 bbdA/L after the first 2 m³ of treated water to remain

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constant afterwards. In both non-inoculated control columns, bbdA numbers in the sand

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fraction were always below the LOQ.

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The distribution profile of MSH1 in the reactors inoculated with free suspended cells was

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determined at the end of the experiment (SI Figure S4). A linear decrease in bbdA numbers

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in the first 40 cm of the column was noted to reach a constant number until the bottom of

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the column. At the top 3.7±0.3 x 107 bbdA/cm³ were found and decreased to 1.5±0.5 x 107

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bbdA/cm³ at 40 cm depth. Total bbdA numbers in the reactors was estimated at 2.3±0.6 x

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1011 bbdA/column after treating 8 m³ water. In the corresponding control column bbdA

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numbers were always below LOQ.

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BAM removal efficiency and MSH1 dynamics in sand filter reactors either inoculated with

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free suspended MSH1 or immobilized MSH1 (Exp 2). To verify results from Exp 1, a second

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experiment (Exp 2) was performed. However, a different procedure of bioaugmentation with

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free suspended cells was used compared to the procedure used in Exp 1. The same high

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density MSH1 cell suspension was applied to the top of the column but instead of a short

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attachment phase of one hour, cells were recirculated over the column for 5 days before

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normal operation was initiated. We hypothesized that this might lead to improved

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attachment and improved performance as MSH1 cells could acclimatize to the sand filter

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environment and hydraulic conditions (shearing).

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In case of inoculation with free suspended MSH1 cells, no difference was observed regarding

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BAM removal compared to Exp 1. BAM removal efficiency was 22% after 44 days of

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operation (time point where Exp 1 was stopped) and 5% at the end of the experiment after

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treating 14 m³ (88 days). In the reactor inoculated with the immobilized MSH1 cells, effluent



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concentrations were below the threshold for the first treated 5 m³ water (30 days). As in Exp

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1, BAM removal was initially 80% but linearly decreased to 20% at 8 m³ (day 44) and 15% at

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the end of Exp 2 (at 16 m³ of treated water, day 88). No BAM removal occurred in the non-

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inoculated control reactors.

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bbdA numbers in the top layer of the filter material and the effluent are shown in SI Figure

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S2 and SI Figure S3, respectively. bbdA numbers in reactors amended with free suspended

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cells followed similar dynamics as in Exp 1. bbdA numbers in the top layer, when flow was

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initiated (day 0) were 8.8±0.3 x 109 bbdA/cm³ and decreased linearly but at a higher rate

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compared to Exp 1, i.e., with a factor 400 in the first 2 m³ of treated water, to reach 2.1±0.8

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x 107 bbdA/cm³ to remain stable afterwards. Compared to Exp 1, the decrease in bbdA

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numbers in the effluent was limited but significant from 5.8±0.5 x 108 until 1.1±0.0 x 108

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bbdA/L in the first 2 m³ of treated water. In the reactor inoculated with immobilized cells,

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bbdA numbers were determined separately in the sand and carrier fraction. The bbdA

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numbers in the carrier were 1.5±0.3 x 109 bbdA/cm³ upon inoculation (0 m3) and decreased

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linearly from 8.8±0.6 x 108 bbdA/cm³ at day 4 (0.7 m³ of treated water) to 1.2±0.1 x 108

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bbdA/cm³ at the end (16 m³ of treated water) of the reactor operation (data not shown). At

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5 m³ of treated water, when BAM started to exceed the EU threshold limit, the total bbdA

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number (carrier and sand together) in the top layer in Exp 2 was 3.6±0.6 x 108 bbdA/cm³ (SI

287

Figure S2). The bbdA numbers in the effluent decreased from 3.4±0.7 x 108 to 1.8±0.1 x 107

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bbdA/L after 2 m³ of treated water and remained constant afterwards (SI Figure S3). bbdA

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numbers in samples taken from the non-inoculated control columns were always below the

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LOQ.

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The distribution profile of MSH1 in the reactors was determined at the experiment (SI

292

Figure S4). In the reactor inoculated with free suspended cells, , a similar linear decrease in

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bbdA numbers in the first 40 cm of the column was noted to reach a constant number until

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the bottom of the column as observed in Exp 1. The bbdA numbers decreased from 5.2±0.2 x

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107 bbdA/cm³ in the top layer until an 1.3±0.7 x 107 bbdA/cm³ at 40 cm depth. Total bbdA

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numbers in the reactor was estimated at 3.3±0.6 x 1011 bbdA/column after treating 16 m³

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water. In the reactor containing immobilized cells, bbdA numbers in the top layer were

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1.4±0.3 x 108 and 2.8±0.2 x 106 bbdA/cm³ material for the carrier and sand fractions,

299

respectively. These numbers decreased to respectively 1.0±0.0 x 107 and 2.8±0.4 x 106 10 ACS Paragon Plus Environment

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bbdA/cm³ material at 40 cm depth and beyond. Total bbdA numbers in the reactor were

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estimated at 8.7±1.6 x 1011 with 4.2±0.7 x 1010 in the sand fraction and 8.3±1.6 x 1011 in the

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carrier fraction indicating that 94±8% of the MSH1 cells were associated with the carrier. In

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the non-inoculated control reactors, bbdA numbers were always below the LOQ.

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MSH1 abundances based on BAM removal rates. The total MSH1 cell numbers in the

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column was only determined at the end of the experiment and data on total MSH1 cell

306

numbers in reactors during the experiment are lacking. Moreover, total MSH1 numbers in

307

Exp 1 in the reactor containing immobilized cells could not be determined (see above). The

308

total BAM-degrading MSH1 cell numbers in the reactors were therefore estimated based on

309

the BAM removal rate (Figure 1) using a previously determined specific BAM-degradation

310

rate of MSH1 cells at 0.2 µg/L BAM21. The specific BAM-degradation rate for freshly grown

311

MSH1 cells in suspended batch conditions was previously determined as 1.1 x 10-12 µg

312

BAM/cell/min21 indicating that minimally 1.2 x 1010 MSH1 cells/column are required to meet

313

the EU threshold limit of 0.1 µg/L BAM. The measured total bbdA numbers at the end of Exp

314

1 and Exp 2 in the reactors exceeded this minimal requirement by a factor 20 in case of

315

bioaugmentation with free suspended cells and 80 in case of bioaugmentation with

316

immobilized cells (Table 2) while BAM removal efficiency was maximally 20% at the end of

317

operation showing that the kinetic parameters derived from freshly grown cells did not

318

describe the kinetics in the reactors well. However, a recently developed kinetic model of

319

BAM-degradation at trace BAM concentrations by sessile MSH1 cells under N and C

320

starvation conditions21, predicted the total number of MSH1 cells in all reactors at the end of

321

the experiment quite well (Table 2) and therefore, this model was used to estimate the

322

number of MSH1 cells in the reactors during the whole operation period (Figure 2). In all

323

reactors, calculated cell numbers decreased logarithmically in function of the treated volume

324

of water and were still significantly decreasing near the end of the experiment indicating

325

that no steady-state was achieved (Figure 2). The calculated total cell loss from the reactors

326

from start until the end of the experiment (at 8 m³ of treated water) was 1.4 fold higher for

327

suspended cells compared to immobilized cells in Exp 1 (Table 2). No significant difference in

328

calculated total cell loss from start until end was observed between suspended and

329

immobilized cells in Exp 2. However, the calculated total loss of cells in the first 2 m³ of

330

treated water was 1.3-fold higher in the reactors amended with suspended cells (suspended 11 ACS Paragon Plus Environment


Page 12 of 27

331

cell strategy Exp 1 and Exp 2) compared to reactors amended with immobilized cells

332

(immobilized cell strategy Exp 1 and Exp 2) (Table 2).

333

The calculated total number of MSH1 cells in the reactors in function of treated volume

334

allowed to determine the total cell loss rate (cells/m³ treated water) and to compare these

335

values with the rate of measured cell loss, i.e., bbdA numbers/m³ effluent. Since the

336

calculated total number of cells lost from the reactors represent cells lost due to volume

337

replacement, shear, grazing and/or decay and the measured lost cells represent cells lost

338

due shear and/or volume replacement, the contribution of shear/volume replacement to

339

total cell loss can be estimated. Results are shown in SI Figure S5. The total cell loss rate was

340

significantly lower for suspended cells compared to immobilized cells in Exp 1. In Exp 2,

341

suspended and immobilized cells did not show a significant difference in total cell loss rate.

342

The measured cell loss rate or shear was at each sampling moment significantly lower than

343

the calculated cell loss rate in all experiments. Right after inoculation, loss rates for

344

suspended cells (Exp 1) were 1 x 1013 cells/m³ of treated water which was largely due to

345

volume replacement/shear since measured cell loss rate and calculated cell loss rate

346

converged. In contrast, the cell loss rate for immobilized cells was about 10-fold lower at the

347

start of reactor operation. In case of suspended cells in Exp 2, initial cell loss rates were in

348

the same order as immobilized cells in Exp 2. Furthermore, at the end of Exp 2, although

349

calculated cell loss rates were not significantly different between the different inoculation

350

strategies (Table 2), differences in measured cell loss rate existed, i.e., differences in

351

contribution of shear/volume replacement to cell loss. In Exp 2, the measured cell loss rate

352

was 3.5-fold higher for suspended compared to immobilized cells. In contrast, in Exp 1, the

353

measured cell loss rate was 2.6-fold lower for suspended cells compared to immobilized cells

354

at the end of operation (Table 2).

355

Discussion

356

In this study, we examined the feasibility of improving BAM removal in sand filter units by

357

bioaugmentation with the BAM degrading Aminobacter sp. MSH1 by adding the strain in an

358

immobilized format using a pilot system approach. The approach was compared to a

359

bioaugmentation approach that was earlier attempted and that was based on inoculation

360

with a free cell suspension22. Pilot scale experiments have the advantage that systems are


Page 13 of 27


361

operated in larger systems under the conditions that are as closely as possible to those

362

expected to occur and to be used in situ but suffer from a limited repeatability and hence

363

limited statistical power due to a lack of sufficient replicates. In this study, this was

364

addressed by repeating the experiment twice in time. Despite the limited replicate number,

365

patterns are clearly emerging regarding both inoculation with free suspended cells (taking

366

into account also previous studies) and immobilized cells and mechanisms of activity loss in

367

the long term.

368

Free cell suspension based bioaugmentation of DWTP sand filters for biological removal of

369

BAM in intake water. Bioaugmentation of pilot scale sand filters in a Danish waterworks was

370

previously demonstrated by applying strain MSH1 in a suspended state22 using an approach

371

similar to the suspended cell procedure applied in Exp 1 in the current study with a similar

372

total amount of added cells. BAM influent concentration were identical and represented

373

typical BAM concentrations in BAM contaminated groundwater31. However, hydraulic

374

properties of the reactors and initial cell density differed substantially between the current

375

study and the study performed at the Danish waterworks22. Despite these differences,

376

similar dynamics in the overall BAM removal rate and MSH1 cell numbers were recorded.

377

Albers et al.22 also reported BAM removal below the EU threshold only in the first 20 days

378

maximal BAM removal rates (75 µg BAM/m³ sand filter/h) were similar to those in the

379

current study (83 µg BAM/m³ sand filter/h). In both studies, BAM removal gradually

380

decreased to reach a BAM removal of 10% and lower and similar final cell densities at the

381

end of the experiment were obtained. Apparently, the time the MSH1 cells reside in the

382

reactor determines the performance of MSH1 more than the hydraulics. This hypothesis is

383

supported by the results obtained with the system inoculated with free suspended cells in

384

Exp 2. The EU threshold limit was exceeded after treating 2 m³ of water while in Exp 1, the

385

reactor performed sufficiently until 3 m³ of treated water. However, the time that the cells

386

resided in the reactors before the BAM effluent concentration exceeded the EU threshold

387

was the same, i.e., 20 days of operation in Exp 1 and 5 days of recirculation plus 14 days of

388

operation in Exp 2. Moreover, both Exp 1 and 2 yielded a MSH1 cell density in the top layer

389

of around 2-3 x 107 cells/cm³. We hypothesize that the time spend in nutrient deprivation

390

and hence starvation resulting in a reduction in BAM-degrading activity in MSH1 biomass is

391

crucial. This is supported by (i) earlier laboratory studies showing that long term N and C



Page 14 of 27

392

limitation affect BAM-degradation in MSH1 in batch and continuous mode due to a

393

reduction of specific BAM degrading activity and/or a reduction in fully active cells21, 23 and

394

(ii) by the observation that in as well this pilot study as in the study of Albers et al. 22, MSH1

395

cell numbers and BAM degradation rate at the end of operation could be explained by BAM-

396

degrading kinetics for starved MSH1 cells21.

397

In exp 2, MSH1 was first precultured on a minimal medium in which BAM was provided as

398

the sole source of carbon and energy. This was performed to ensure a starting culture that

399

mineralizes BAM for further propagation in R2B containing BAM to suitable inoculum

400

densities, since we noted that MSH1 can lose the part of the BAM degradation pathway that

401

degrades DCBA further to CO2. Final propagation was however similar in both experiments

402

and therefore cell physiology should be the same in both experiments and differences in cell

403

attachment capacity between both experiments are very unlikely. Also final MSH1 cell

404

numbers were not significantly different in the reactor inoculated with the free suspended

405

cells in Exp 1 and 2 and BAM-removal rates were the same, strongly suggesting that the

406

initial preculturing step using BAM as sole C-source and the possible higher number of MSH1

407

cells that can mineralize BAM did not affect final cell numbers and performance of MSH1.

408

AOC has been identified as the main carbon and energy source for MSH1 when treating

409

water contaminated with BAM at trace concentrations21,

410

current study at approximately 20 μg/L which is a usual concentration in drinking water32.

411

Therefore, the ability to mineralize BAM and use BAM for growth, is not a selective

412

advantage in our reactors. Whether a MSH1 inoculum with low incidence of BAM-

413

mineralizing cells can lead to the production of certain metabolites like DCBA needs further

414

investigation. Currently, DCBA is the only known metabolite in the BAM-mineralization

415

pathway of MSH117. BAM can be mineralized at trace concentrations39 and DCBA was not

416

detected in the study of Albers et al.22. Recently, Vandermaesen et al.40 showed that the

417

indigenous microbial community in several sand filters showed mineralization of DCBA.

418

Therefore, when BAM is only transformed to DCBA by MSH1, the sand filter community

419

might be able to mineralize DCBA.

420

Immobilized cell strategy improves bioaugmentation compared to using suspended cells

421

by reducing the shear rate. BAM removal below the EU threshold was three times longer

422

using freshly produced immobilized MSH1 cells (Exp 1) and two times longer when the

22

and AOC was estimated in the


Page 15 of 27


423

carriers were stored at 4°C for another 6 months (Exp 2) compared to suspended cells (Exp

424

1). The difference between immobilized cells and suspended cells regarding the time period

425

to reach the EU threshold concentration appears in the first place explained by the fact that

426

immobilization reduces considerably the initial loss of unattached cells due to volume

427

replacement or poorly attached cells by shear, compared to free suspended cells. This can be

428

clearly deduced from the measured and calculated cell losses during the experiment. When

429

suspended cells were added in Exp 1, initial cell loss due to shear was 9±1 x 1012 cells/m³ of

430

treated water which was reduced 10-20 fold when cells were immobilized. This led to a clear

431

difference in cell loss from the reactors after treating 2 m³ water with 85% of the inoculum

432

lost when cells were immobilized and 90% when cells were suspended. This reduction in cell

433

loss can be attributed to their incorporation in the alginate matrix since carriers without

434

alginate showed a release of nearly 100% of MSH1 compared to only 0.01% when alginate

435

was used (A. Lapanje, unpublished results). After 8 m³ of treated water, the total cell loss

436

was 90% for immobilized cells in both Exp 1 and 2 and 99% (in Exp 1) and 95% (in Exp 2) for

437

suspended cells. In Exp 2, after 16 m³ the total amount of lost cells (around 98%) from what

438

was added was not significantly different between the reactors inoculated with suspended

439

and immobilized cells (Table 2). However, at the end of operation, a 2.5±0.5 fold higher cell

440

density was found in the reactor containing immobilized cells compared to this inoculated

441

with suspended cells. Losses due to shear stress at a later stage for immobilized cells became

442

minimal (5-6% of total loss) compared to suspended cells with 1% of total loss due to shear

443

although a higher shear stress is expected to occur in the reactor containing immobilized

444

cells. The presence of the carriers in the column will reduce the volume through which the

445

water flows and hence doubles the pore water flux in the column compared to a reactor that

446

only contains the sand and hence will double the shear stress in reactors containing the

447

carrier. Final total cell loss rates between suspended and immobilized were not significantly

448

different. Furthermore, immobilized cells are known to be less prone to grazing when

449

located in pores (

Biocarriers Improve Bioaugmentation Efficiency of a Rapid Sand Filter

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