Surface Colonization and Activity of the 2,6-Dichlorobenzamide (BAM

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Surface colonization and activity of the 2, 6-dichlorobenzamide (BAM) degrading Aminobacter sp. strain MSH1 at macro- and micropollutant BAM concentrations Aswini Sekhar, Benjamin Horemans, Jens Aamand, Sebastian R. Sorensen, Lynn Vanhaecke, Julie Vanden Bussche, Johan Hofkens, and Dirk Springael Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01978 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 18, 2016

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Environmental Science & Technology

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Surface colonization and activity of the 2, 6-dichlorobenzamide (BAM) degrading

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Aminobacter sp. strain MSH1 at macro- and micropollutant BAM concentrations

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Aswini Sekhara†, Benjamin Horemansa†, Jens Aamandb, Sebastian R. Sørensenb, Lynn

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Vanhaekec, Julie Vanden Busschec, Johan Hofkensd and Dirk Springaela*

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a

8

Leuven, Belgium

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b

Division of Soil and Water Management, KU Leuven, Kasteelpark Arenberg, BE-3001

Department of Geochemistry, Geological Survey of Greenland and Denmark (GEUS), DK-

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1350 Copenhagen, Denmark

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c

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UGent, BE-9000 Ghent, Belgium

13

d

14

†

Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis,

Molecular Imaging and Photonics, KU Leuven, BE-3001 Leuven, Belgium A.S and B.H. contributed equally to this paper and are both first authors.

15 16

*Corresponding author:

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Dirk Springael

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Division of Soil and Water Management

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KU Leuven

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Kasteelpark Arenberg 20

21

BE-3001 Heverlee

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Belgium

23

Tel: ++32 16 32 16 04

24

Fax: ++32 16 32 19 97

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E-mail: [email protected]

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Keywords: Surface colonization, micropollutant biodegradation, 2,6–dichlorobenzamide

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(BAM), drinking water production

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Abstract

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Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM)

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as C and N source and is a potential catalyst for biotreatment of BAM contaminated

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groundwater in filtration units of drinking water treatment plants (DWTPs). The oligotrophic

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environment of DWTPs including trace pollutant concentrations and the high flow rates

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impose challenges for micropollutant biodegradation in DWTPs. To understand how trace

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BAM concentrations affect MSH1 surface colonization and BAM degrading activity, MSH1

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was cultivated in flow channels fed continuously with BAM macro- and microconcentrations

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in a N and C limiting medium. At all BAM concentrations, MSH1 colonized the flow

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channel. BAM degradation efficiencies were concentration dependent ranging between 70

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and 95%. Similarly, BAM concentration affected surface colonization but at 100 µg/L BAM

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and lower, colonization was similar to this in systems without BAM, suggesting that

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assimilable organic carbon and/or nitrogen other than those supplied by BAM sustained

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colonization at BAM microconcentrations. Comparison of specific BAM degradation rates in

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flow channels and in cultures of suspended freshly grown cells indicated that starvation

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conditions in flow channels receiving BAM microconcentrations resulted into MSH1 biomass

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with 10-100 times reduced BAM degrading activity and provided a kinetic model for

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predicting BAM degradation under continuous C and N starvation.

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Introduction

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Stringent European pesticide residue threshold concentrations are set for drinking

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water, i.e., 0.1 µg/L for individual compounds1. 2,6-dichlorobenzamide (BAM) is a

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transformation product of the widely used herbicide dichlobenil (2,6-dichlorobenzonitrile)2-5

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and is a common and highly persistent groundwater pollutant5, 6. BAM groundwater

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concentrations are in the micropollutant range (i.e, ng – µg/L scale) but often still exceed the

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EU drinking water threshold concentration resulting in the costly closure of extraction wells 2 ACS Paragon Plus Environment

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or in expensive measures such as activated carbon filtration in DWTPs to meet drinking

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water standards7. Alternative measures to treat BAM contaminated groundwater are of

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interest8.

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Aminobacter sp. MSH1 is an aerobic soil isolate that uses dichlobenil and BAM as

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sole source of carbon, nitrogen and energy6, 9. The strain is a gram-negative motile rod that

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can mineralize BAM at the micropollutant concentrations encountered in contaminated

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groundwater10 and a potential catalyst in alternative biological approaches for removing

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BAM in DWTPs11. One approach is bioaugmentation of filtration units employed in DWTPs

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such as sand filter units used for manganese and iron oxide removal as proposed by Albers et

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al.12. In DWTP filtration units, MSH1 should colonize the solid substratum while maintaining

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BAM degrading activity. DWTPs are, however, characterized by (i) micropollutant BAM

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concentrations (µg/L-ng/L range), (ii) the oligotrophic conditions and (iii) short hydraulic

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retention times and high flow rates, that imply specific challenges for BAM biodegradation in

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continuous systems. The micropollutant BAM concentrations and the oligotrophic

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enviroment can impact cell growth, gene expression and enzymatic substrate recognition8. In

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case of biofilms, diffusion of BAM into and through the biofilm might be an extra constraint

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on degradation efficiency at low concentrations12, 13. Short hydraulic retention times lead to

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short contact times between the microbial catalyst and the pesticide6, 14-16, while high flow

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rates invoke shear stress and cell loss17-19. Knowledge on how those constraints affect

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colonization of substrata and the degradation activity of micropollutant degrading organisms

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under continuous conditions is however scarce.

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To improve our understanding of the impact of the oligotrophic environment of

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DWTPs on the long term establishment and activity of micropollutant degrading organisms

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for bioremediation of polluted intake water in DWTPs, we examined how trace

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concentrations of BAM affect the colonization of a surface by Aminobacter sp. MSH1 and its

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BAM degrading activity under continuous regimes. To this end, MSH1 was cultured in 3 ACS Paragon Plus Environment


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continuous flow channels irrigated with a C and N limiting minimal medium containing

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BAM at macro- and micropollutant concentrations until a nominal concentration as low as 1

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µg/L. BAM was the only added C and N source in the medium and hence defined the

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nominal N and C content of the medium. The applied water flux was 0.6 m/h and the

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hydraulic retention time 3 min which is comparable to those employed in DWTP filtration

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units. Surface colonization was analyzed by confocal laser scanning microscopy (CLSM) and

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degradation rates and cell yields between the different concentrations were compared.

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Comparison between specific BAM degradation rates in suspended batch conditions and in

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the flow channels allowed for conclusions about the effect of sessile growth and long term C

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and N-limiting conditions on the BAM degrading activity of MSH1 in flow-through

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purification systems in DWTPs.

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Materials and methods

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Bacteria and culture conditions. Aminobacter sp. MSH16 was grown on R2A or its broth

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version R2B20 containing 10 mg/L BAM. Aminobacter sp. MSH1-GFP is a green fluorescent

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protein (GFP) labeled and kanamycin (Km) resistant variant of MSH1 constructed by

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introduction of the GFP-2X-miniTn5Km gene cassette21. Mutant M1a100g is a spontaneous

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mutant of MSH1-GFP deficient in converting BAM to 2,6-dichlorobenzoic acid (DCBA)22.

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Flow channel experiments. Experiments were performed at 25oC in three channel flow

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chambers (©Biocentrum DTU, Denmark) with channel dimensions of 1x4x40 mm23. The

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flow chamber is made from polycarbonate and contain a microscope borosilicate glass

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coverslip as substratum (Menzel-Gläser, Germany)23. The procedure for assembly,

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sterilization and inoculation of the flow chamber setup was described previously23. All

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elements of the flow chamber setup are connected using Viton® rubber tubing. After

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sterilization by injecting 0.5% sodium hypochlorite (Acros Organics, Geel, Belgium) and 4 ACS Paragon Plus Environment

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leaving it for 3 h, the channels were rinsed with sterile ultrapure water (Milli-Q®).

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MSH1/MSH1-GFP/M1a100g inocula grown in R2B containing 10 mg/L BAM at 25oC and

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harvested at an optical density at 600 nm (OD600) of 0.6, were washed three times and

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resuspended in 0.9% NaCl at an OD600 of 0.7. The flow chamber was turned upside down

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and 250 µL inoculum was injected. After 1 h, the flow chamber was turned and the medium

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flow initiated at 3.4 mL/h1 (hydraulic retention time of 3 min) using a peristaltic pump

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(Watson Marlow 205S). The feed media were mineral salt (MS) medium24 containing

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different nominal concentrations of BAM, i.e., 1 µg/L, 10 µg/L, 100 µg/L, 1000 µg/L, 10

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mg/L, 60 mg/L and 100 mg/L or without BAM. BAM concentrations in the 1 to 100 mg/L

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range were designated macropollutant concentrations while concentrations in the 1 to 100

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µg/L range micropollutant concentrations. Each concentration was tested in triplicate. Three

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experiments were performed designated as experiments 1, 2 and 3. Experiment 1 used wild

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type MSH1 as inoculum and tested all above mentioned BAM concentrations. Experiment 2

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used MSH1-GFP as inoculum and tested BAM concentrations of 1 mg/L, 1 µg/L and 0 µg/L.

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Experiment 3 used MSH1-GFP and M1a100g as inocula and tested BAM concentrations of 1

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mg/L, 1 µg/L, and 0 µg/L. Non-inoculated abiotic controls were operated in duplicate for the

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BAM concentrations 1 µg/L (in experiments 1, 2 and 3), 10 µg/L (in experiment 1), 100 µg/L

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(in experiment 1) and 1 mg/L (in experiment 3). One ml effluent samples for BAM analysis

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were collected twice every week in tubes containing 5 µl 5 M HCl to stop microbial activity.

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Precise influent BAM concentrations were determined upon replacing the feed and at the end

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of the experiments. Fifty µL effluent samples were regularly taken for enumerating colony

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forming units (CFU) or occasional microscopic inspection.

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Determination of BAM concentrations. BAM concentrations higher than 100 µg/L

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were determined by UHPLC as described22 using an injection volume of 20 µL. Limits of

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detection (LOD) and quantification (LOQ) were 56 µg/L and 170 µg/L, respectively. 5 ACS Paragon Plus Environment


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UHPLC-MS/MS (Thermo Scientific, San José, USA) consisting of an Accela pumping

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system coupled with a triple quadrupole mass analyzer (TSQ Vantage), was used to

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determine BAM concentrations below 100 µg/L as described in SI. LOD and LOQ for BAM

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were 1 ng/L and 5 ng/L, respectively.

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CLSM and image analysis. CLSM analysis was performed at the end of the

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experiment when effluent BAM concentrations were constant and hence BAM degradation at

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steady state. Cells were stained by injecting 250 µL of a 100 nM SYTO® 62 Red Fluorescent

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Nucleic Acid stain (Molecular Probes™, ThermoFischer Scientific) solution into the

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channels. When appropriate, and after SYTO® 62 staining (one hour), the biomass was

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stained (one hour) with 250 µl of 500 µg/mL of Concanavalin A-TRITC (Molecular

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Probes™, ThermoFischer Scientific) targeting EPS mannopyranosyl and glucopyranosyl

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residues. The channels were rinsed with 500 µL 0.9% NaCl and analyzed on an Olympus

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IX70 inverted microscope with a Fluoview FV500 confocal scanning unit. ConA-TRITC,

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GFP and SYTO® 62 signals were collected sequentially. GFP was visualized using the 488

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nm laser for excitation in combination with a 494–520 nm band pass emission filter, SYTO®

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62 using the 633 nm laser combined with a 660 nm long pass emission filter and ConA-

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TRITC using the 560 nm laser combined with a 572–620 nm band pass emission filter.

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Images were captured at front, middle and back of the channels at three positions at 1 µm

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increments (512x512pixel frame; 0.414 µm pixel size) with a 60x oil immersion UPL SAPO

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x60/1.35 objective (Olympus, Japan). Imaris 7.2 software (Bitplane, USA) generated

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simulated projections and sections through the CLSM images. MSH1 and EPS biovolumes

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were calculated using COMSTAT25 with details provided in SI.

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Enumeration of colony forming units (CFU) and microscopic analysis. Samples

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for counting CFUs of attached inoculum cells in the flow channels were taken at the end of 6 ACS Paragon Plus Environment

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the experiment by rinsing with 300 µL 0.9% NaCl for 5 min. Samples were thouroughly

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vortexed and pipetted up and down to break aggregates before serial dilution in 0.9% NaCl.

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Dilutions were plated on R2A for enumerating MSH1 or on both R2A and R2A

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supplemented with 50 mg/L Km for enumerating MSH-GFP and M1a100g. Differences in

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CFU numbers between R2A plates with and without Km would indicate the presence of

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contaminant organisms in the flow channels since R2A with Km allows the selective growth

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of the Km resistant MSH1-GFP and M1a100g strains. However, no differences were

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observed in CFU counts on R2A and R2A Km plates. Effluent samples were identically

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treated and plated on R2A with 50 mg/L Km since plating of effluent samples on R2A

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yielded contamination with bacteria that colonized the exterior of the effluent exit. In all

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cases, CFUs were counted after 2-8 days incubation at 25°C. Selected effluent samples were

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examined using an upright epifluorescence microscope (Olymbus BX51) with filter set U-

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M41001, composed of a 461-500 nm excitation filter and a 521-560 nm barrier filter with a

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splitting wavelength of 505 nm. The samples were treated with SYTO® 62 as described

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above and 10 µl examined at 100X magnification.

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BAM degradation in suspended batch systems containing freshly grown MSH1-GFP

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cells. Batch degradation assays were performed in triplicate in 100 ml Erlenmeyer flasks

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containing 25 ml MS medium with nominal BAM concentrations of 1, 5, 10, 50, 100, 500 or

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1000 µg/L. Three abiotic controls without cells were included for all BAM concentrations.

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Actual concentrations were measured before inoculation. MSH1-GFP grown in R2B

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containing 10 mg/L BAM at 25oC, was harvested at an OD at 600 nm of 0.6, washed and

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resuspended in 10 mM MgSO4 at an OD at 600 nm of 0.25. Erlenmeyers with concentrations

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of 1 to 500 µg/L BAM were inoculated at 2.5 x 107 cells per mL and Erlenmeyers containing

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1000 µg/L at 2.5 x 108 cells per mL. Erlenmeyers were incubated at 25°C while shaking (150

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rpm). One mL samples were taken every 15 minutes during 3 hours in tubes containing 10 µl

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37% HCl-solution and BAM concentrations determined as described above.

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Calculation of biomass, cell yields, AOC content and BAM (specific) degradation rates

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and kinetic parameters in flow channels and/or suspended batch cultures. Overall BAM

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degradation rate, average total relative MSH1 biovolumes, number of MSH1 cells based on

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biovolumes, specific BAM degradation rate (per CFU and MSH1 biovolume), cell yields in

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flow channels, kinetic parameters in batch cultures as well as assimilable organic carbon

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(AOC) estimations were calculated as reported in SI.

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Statistics. One-way analysis of variance (ANOVA) (significance level of 0.05) was used to

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compare values of total biovolumes in the flow channels, total CFU numbers in the flow

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channels, specific degradation rates in the flow channnels (per MSH1 cell biovolume and per

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MSH1 CFU), total number of detached cells, total number of produced cells and cell yields

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between setups that received different BAM concentrations within the same experiment. All

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data were log transformed prior to statistical analysis. The unpaired two tailed Student’s t-test

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was used to test significant differences. (significance level 0.05) between values of

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MSH1/MSH1-GFP/M1a100g

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GFP/M1a100g cell numbers based on biovolume estimations within the same setup of an

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experiment.

cell

numbers

counted

by

CFU

and

MSH1/MSH1-

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Results

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BAM macro- and microconcentrations. BAM removal % at macropollutant concentrations

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reached steady state at around 50 days for the nominal concentrations of 10, 60 and 100 mg/L

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BAM and around 20 days for 1 mg/L BAM (Figure 1A). For micropollutant BAM

BAM degradation activity and colonisation profile of MSH1 in flow channels fed with


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concentrations, more or less steady state degradation was already achieved at the first

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sampling time point, i.e., at 10 days (Figure 1B). At the end of the experiment, the BAM

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removal was 80 to 88% for all nominal BAM concentrations except for 1 µg/L, 10 mg/L and

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100 mg/L (Table 1) showing removal of 70±2%, 95± 0% and 94±3%, respectively. BAM

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was not removed in non-inoculated flow channels (SI Figure S1).

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CLSM analysis at the end of the experiment showed high biomass with macrocolonies

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in setups receiving macropollutant concentrations (SI Figure S2A). Biomass decreased from

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the front to the back of the channel (SI Figure S3). In systems receiving BAM

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microconcentrations and no BAM, microcolonies and individual cells evenly spread along the

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channel (SI Figure S2B), with no apparent difference in biomass and structure between

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systems receiving and those not receiving BAM (SI Figure S3). Both at BAM macro- and

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microconcentrations, individual cells were observed several micrometers away from the

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surface attached biomass indicating the presence of EPS (data not shown). No growth

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occurred in abiotic controls (SI Figure S2B).

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MSH1 cell numbers based on biovolume (per flow channel) and CFU in the

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inoculated flow channels at the end of the experiment were not significantly different

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between flow channels receiving nominal BAM concentrations of 100, 60, and 10 mg/L

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BAM but were lower in flow channels receiving 1 mg/L BAM (approximately 10 fold) and

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100 µg/L BAM and lower concentrations (20 fold) (Table 1). MSH1 biovolumes and CFU in

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flow channels fed with microconcentrations BAM, however, were similar to those recorded

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in channels that did not receive BAM (Table 1). Particular observations were done regarding

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the CFU counting of biomass extracted from the flow channels. CFU development on R2B

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occurred slower for cells extracted from flow channels fed with BAM micropollutant

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concentrations (one week) compared to those from macropollutant concentration fed channels

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(2 days). Moreover, enumeration of MSH1 cells based on biovolume and CFU were often

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significantly different. At nominal BAM concentrations higher than 10 mg/L, MSH1 cell 9 ACS Paragon Plus Environment


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numbers based on CFU numbers were higher than those based on biovolumes while the

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opposite was true at feed nominal concentrations of 100 µg/L and lower (Table 1). Hence,

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calculated specific degradation rates depended on the enumeration method. In general,

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overall and specific degradation rates decreased with decreasing BAM concentration (Table

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1).

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Detailed investigation of MSH1 colonisation pattern in systems receiving 1 mg/L and 1

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µg/L BAM. The presence of individual cells above the main biofilm suggested the

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production of EPS. Moreover, no difference existed between MSH1 cell numbers in flow

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channels that received BAM micropollutant concentrations and those that did not receive

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BAM, suggesting that at micropollutant concentrations BAM does not govern MSH1 growth

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in the channels. To examine both hypotheses, MSH1-GFP was grown in systems fed with

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either 1 mg/L BAM, 1 µg/L BAM and no BAM, and in addition to the parameters measured

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in the previous experiment, EPS was visualized by appropriate staining and CFU were

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determined regularly in the effluent, as well as in the flow channels at the start and the end of

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the experiment to calculate cell yields. The use of MSH1-GFP allowed improved

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visualization of individual MSH1 cells. Non-inoculated flow channels receiving 1 µg/L BAM

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were operated as abiotic controls.

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Overall, BAM removal was as in experiment 1 (SI Figure S4, Table 1). The removal

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of BAM at the end of the experiment was however higher for the 1 µg/L BAM feed compared

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to experiment 1 (i.e., 86±3% instead of 70±2%) but the actual BAM concentration in the feed

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deviated from the nominal concentration of 1.0 µg/L and was 1.5 µg/L (Table 1). The amount

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and mode of MSH1-GFP surface colonization was identical to those in experiment 1 with

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small colonies and single cells for feeds of 1 µg/L BAM and no BAM (Figure 2A). In all

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systems, GFP production varied between cells. Counter staining with Syto62 showed that as

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observed in biofilms with other bacterial strains26, cells were either Syto62 or GFP stained

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with non-GFP producing cells (Figure 2A) making a substantial fraction of the cells 10 ACS Paragon Plus Environment

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(26±20%) and no significant difference between the feed conditions (SI Figure S5). No

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growth occurred in the abiotic systems and hence Syto62 stained cells are likely true MSH1

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cells and not contaminant cells. GFP-production though was weaker in cells in systems

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receiving 1 µg/L BAM and no BAM compared with those fed with 1 mg/L BAM (data not

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shown). Staining with ConA confirmed the presence of EPS polysaccharides loosely

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structured above the main biomass up to 50 µm in height (Figure 2B) and containing GFP

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and non-GFP producing single cells (Figure 2C). Volume of EPS per MSH1 cell (based on

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biovolume) was the same for all feeding conditions (SI Figure S6). As in experiment 1, total

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MSH1-GFP biovolumes and CFU numbers in the flow channels depended on the BAM feed

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concentration with no differences between the feed of 1 µg/L BAM and without BAM (Table

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1). In the latter conditions, cell numbers based on CFU were lower than those based on

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biovolumes. As in experiment 1, more time was needed for cells extracted from channels

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receiving the 1 µg/L BAM feed and the feed without BAM to form CFU on R2B Km

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compared to cells from the 1 mg/L feed. The actual concentration of 1.5 µg/L BAM in the

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feed instead of 1 µg/L (as in the first experiment) increased the overall and specific BAM

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degradation rate based on biovolume with a factor 3 (Table 1).

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Effluent CFU numbers reached steady state 24 hours after inoculation showing

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1.0±3.0 x 107 CFU/mL for channels receiving 1 mg/L, 1.1±1.0 x 106 CFU/mL for systems

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fed with 1 µg/L BAM, and 3.0±2.0 x 105 CFU/mL for channels receiving no BAM.

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Interestingly, CFU formation from effluents from channels fed with 1 µg/L and no BAM

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became slower over time and at steady state, as the cells extracted from the flow channel,

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took more than one week while those from channels fed with 1 mg/L BAM needed maximum

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2 to 3 days. Microscopic inspection showed the occurrence of cell aggregates of 3 to 10 cells

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among single cells in the effluent especially in samples from the systems receiving 1 µg/L

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BAM and no BAM (data not shown). The total number of CFU produced in the channels 11 ACS Paragon Plus Environment


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receiving 1 mg/L BAM was significantly more than in those fed with 1 µg/L BAM (3 times

296

higher) and MS without BAM (10 times higher) (SI Table S1). Estimated yields on BAM

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were around 100 times lower for channels receiving 1 mg/L BAM compared to those

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receiving 1 µg/L BAM (SI Table S1), suggesting that indeed at micropollutant

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concentrations, other carbon sources than BAM determined MSH1 growth.

300 301

Behavior of a BAM degradation defective mutant of MSH1 in flow channels. To acquire

302

further evidence that in systems fed with 1 µg/L BAM, BAM did not control growth of

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MSH1 and that no BAM was retained in the systems by sorption to EPS or to cells, surface

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colonisation by MSH1-GFP and mutant M1a100g defective in BAM degradation, in flow

305

channels fed with either 1 mg/L BAM, 1 µg/L BAM and no BAM was compared. Non-

306

inoculated sytems were operated as abiotic controls. MSH1-GFP behaved as in experiment 2.

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Strain M1a100g did not show BAM removal neither when fed with 1 mg/L or 1 µg/L (actual

308

concentration was 2.2 µg/L) (SI Figure S7 and Table 1). M1a100g EPS production, biomass

309

size and colonization pattern (data not shown) were identical to those recorded for MSH1-

310

GFP receiving 1 µg/L or no BAM. No difference in break-through and retention of BAM was

311

observed between abiotic systems and systems inoculated with M1a100g indicating that

312

sorption of BAM to MSH1 biomass does not contribute to BAM removal by MSH1 in the

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flow systems (SI Figure S1 and S7). At the end of the experiment, biovolumes of mutant

314

M1a100g receiving 1 mg/L BAM were four times less compared to those of MSH1-GFP fed

315

with 1 mg/L BAM but were only slightly lower than those of MSH1-GFP receiving 1 µg/L

316

BAM or no BAM (Table 1). Similarly, significant differences existed in CFU number of

317

MSH1-GFP and M1a100g in channels fed with 1 mg/L BAM but not in those receiving 1

318

µg/L or no BAM (SI Table S1). Unexpectedly, MSH1-GFP CFU numbers in the flow

319

channels were around 100 times lower compared to experiments 1 and 2 for the feeds

320

containing 1 µg/L BAM or no BAM, and hence specific BAM degradation rates per CFU 12 ACS Paragon Plus Environment

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differed between the experiments for the feeds containing 1 µg/L BAM. In contrast, MSH1-

322

GFP biovolumes and overall and specific BAM degradation rates expressed per biovolume

323

were identical for all three experiments (Table 1).

324

The inability of M1a100g to metabolize BAM did not influence the CFU number in

325

the effluent even at a feed of 1 mg/L (SI Table S1). MSH1-GFP and M1a100g showed

326

similar numbers of CFU (SI Table S1) in the effluent of flow channels receiving 1 mg/L

327

BAM and 1 µg/L BAM for the entire duration of the experiment as well as for the total

328

number of cells produced (SI Table S1). In channels fed with MS medium without BAM,

329

M1a100g CFU numbers in the effluent and total CFU produced were ten fold less compared

330

to MSH1-GFP numbers, while biomass was the same (Tables 1 and SI Table S1).

331 332

BAM degradation by MSH1-GFP in batch systems with suspended fresh cells. Specific

333

degradation rates in suspension plotted against initial BAM concentration (Figure 3 and Table

334

2) followed Monod kinetics showing decreasing degradation rates with decreasing

335

concentration. No BAM removal was observed in the abiotic controls. Using the Monod

336

model, Vmax [µg/cell/min] and Ks (µg/L) were estimated at 5.4 ± 0.2 x10-11 µg

337

BAM/cell/min (2.8 ± 0.1 x10-13 µmol/cell/min) and 99.6 ± 13.7 µg/L (0.52 ± 0.07 µM),

338

respectively. Estimated specific BAM degradation rates in suspension were a factor 10-100

339

higher than those estimated for the flow chamber experiments based on MSH1-GFP cell

340

biovolumes and a factor 10-25 higher than those estimated in the flow channels based on

341

CFU counts (Table 2).

342 343

Discussion

344

This study relates to a bioaugmentation strategy that uses strain MSH1 in biofiltration units

345

for treating intake water contaminated with BAM micropollutant concentrations in DWTPs.

346

In such systems, strain MSH1 is expected to (i) attach to the solid substratum of the unit, (ii) 13 ACS Paragon Plus Environment


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347

maintain biomass by growth to compensate for losses due to shear and decay and (iii)

348

maintain BAM degrading activity despite the conditions of oligotrophy, trace concentrations

349

of selective substrate and the high fluxes and shearing forces due to high water flow rates

350

expected to occur in DWTP filter units. In this paper, we examined how oligotrophic

351

conditions and low substrate concentrations affect the establishment of active MSH1 cells in

352

such systems focussing on N and C limitation, the two main elements provided by BAM by

353

means of flow chamber experiments. Hydraulic parameters were kep constant and were near

354

those encountered in DWTP filtration units. For instance, in sand filters used in DWTPs, that

355

have been proposed as possible units for bioaugmentation, fluxes range from 0.4 m/h for slow

356

and 4-20 m/h for rapid sand filtration27. The flux in our system was set at 0.6 m/h. Assuming

357

that cells and microcolonies are shaped similarly in flow channels and in sand filters,

358

hydraulic properties and the shear forces on biofilms in the flow channels were as such

359

similar to those in slow sandfilters. The setup allows visualisation of the surface colonisation

360

and provides insights into the small-scale processes within a biofilter operating at realistic

361

conditions.

362

Surface colonization of MSH1 at BAM micropollutant concentrations. Our results show

363

that MSH1 colonizes the flow channels even at BAM concentrations as low as 1 µg/L (5.2

364

nM) under continuous flow conditions. This is the first study showing colonisation of a solid

365

surface by a xenobiotic utilizing/degrading microoorganism receiving the xenobiotic at

366

environmental relevant microconcentrations of around 1 µg/L (i.e., 10 times the treshold limit

367

for drinking water) under continuous mode mimicking the conditions in DWTP biofilters.

368

Horemans et al.28, showed surface colonisation by a linuron degrading consortium at 10 µg/L

369

(40.2 nM) linuron using the same flow channel setup. Successful colonisation will first

370

depend on initial cell attachment. Attachment and colonization can depend on the material

371

used as solid substratum. In the flow channel experiments, glass was employed as the solid

372

substratum. Glass is a silica-based material as is the material employed in sand filters and 14 ACS Paragon Plus Environment

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373

appears a suitable substratum for initial attachment of and further colonisation by MSH1

374

under hydraulic and shear conditions similar to those found in DWTP filtration units such as

375

sand filters. Previously, MSH1 attached to material in sand columns at 5–50% of the added

376

biomass29, which is similar to the 13% attachment in our study corresponding to 8 x 104 cells

377

per µm². After attachment, strain MSH1 clearly grows and maintains its biomass even at 1

378

µg/L BAM thereby compensating for cell loss which was estimated as 2.4 x 106 cells/h at 1

379

µg/L BAM. Surface colonisation at micropollutant concentrations differed substantially from

380

this at macropollutant concentrations. Small colonies and single cells were observed

381

reminiscent of the low carbon and nitrogen content of the medium. A similar colonisation

382

pattern was observed for the linuron degrading consortium fed at 10 µg/L linuron28.

383

Previously, MSH1 was shown to mineralize BAM at micropollutant concentrations as low as

384

1.9 µg/L10 in suspended batch systems. However, BAM did not govern the growth in

385

channels at BAM micropollutant concentrations. We suggest that MSH1 biomass formation

386

at micropollutant BAM concentrations is mainly supported by assimilable organic carbon

387

(AOC) sources as well as unknown trace N (organic) sources present in the minimal medium

388

or released from the flow chamber setup which included plastic and rubber materials. Based

389

on the cell yields in setups fed without BAM, AOC was estimated to be around 48±37 µg

390

C/L (see SI Table S1) which is in the order of reported AOC contents in potable water in

391

Flanders (Belgium)30 and in synthetic minimal media in terms of total carbon31 while

392

concentrations of unknown N sources would be then at least around 5 µg N/L. Standard

393

laboratory mineral media might even contain up to 1 mg/L of contaminating AOC32. Cell

394

yields based on CFU did not take into account cell aggregation which indicates that actual

395

AOC and N contents were a factor 5 to 10 higher. This is a factor 250-1000 higher than AOC

396

and 25-100 times higher than N which MSH1 derives from BAM concentrations found in

397

groundwater (0.1 to 2 µg/L) and implies that even at a concentration of 100 µg/L BAM, other

398

AOC and N is in surplus. The importance of growth on a surplus of residual AOC while 15 ACS Paragon Plus Environment


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399

degrading pollutants was reported previously for a dichlorophenol degrading Pseudomonas

400

sp.31 and is considered deterministic when assessing growth yields at micropollutant

401

concentrations in batch systems32.

402

Interestingly, cell estimations based on biovolume were higher (10 to 100 fold) than CFU

403

based estimations for the lower BAM concentrations (100 µg/L and below) but lower (10

404

fold) for the higher concentrations (60 mg/L and above). In case of 10 mg/L and 1 mg/L feed,

405

cell numbers based on CFU and biovolume were identical. The discrepancy at the highest

406

BAM concentrations is inherent to CLSM where cell acquisition in biofilm top layers that

407

extend more than 30 µm above the surface is less accurate. For the lower concentrations, this

408

discrepancy is at least partially explained by cell aggregation. Apparently cells from systems

409

receiving BAM at microconcentrations and no BAM sticked more together than those from

410

systems receiving 1 mg/L BAM since CFU numbers and MSH1 biovolumes agreed at 1 mg/L

411

feed and more single cells were seen under the microscope. Furthermore, CFU formed

412

progressively slower in time from effluent cells sampled from channels fed with BAM

413

microconcentrations and at steady state, CFU formation from samples from channels

414

receiving BAM microconcentratons was much slower than from samples from BAM

415

macroconcentration fed systems. We hypothesize that especially in the channels fed with

416

BAM microconcentrations, not all sessile cells are as metabolically active and/or that a large

417

fraction of cells is dormant and rather represent viable but not culturable cells (VBNC). Also

418

Sjøholm et al.33 observed that prolonged incubation in C- and N-free mineral solution in

419

batch conditions affected the growth resumption of MSH1 on agar although cell membrane

420

functionality was maintained indicating intact cells. Interestingly, at the single cell level, GFP

421

fluorescence by attached MSH1-GFP cells was less when fed with 1 µg/L BAM compared to

422

the 1 mg/L feed indicating a lower activity. Moreover, as discussed below, specific BAM

423

degradaton rates were 100 times lower (based on biovolume measurements) in the flow

424

channels compared to suspended fresh cells. The microconcentration situation might be 16 ACS Paragon Plus Environment

Page 17 of 32


425

related to starvation conditions33 and bacterial cells can become VBNC under nutrient

426

starvation34. The observation that with higher feed BAM concentration CFU formation was

427

not affected indicates that cells became more metabolically active with more available carbon

428

source, in this case BAM.

429

Another observation was that the amount of detached cells did not largely differ (less than

430

one order) between the 1 mg/L, 1 µg/L and no BAM feed concentrations. Indeed, based on

431

the effluent cell numbers at steady state, we calculated that in channels receiving 1 mg/L, 1

432

µg/L and no BAM in experiment 2, the number of planktonic cells were respectively 0.012,

433

0.037 and 0.006% of the sessile biomass (without taking into account cell aggregation).

434

Apparently, starved MSH1 cells were more prone to detachment. Increased detachement

435

upon starvation has been reported and might be due to increased sensitivity to shear stress but

436

also matrix degradation or cell lysis35, 36.

437

Finally, strain MSH1 produces EPS. This EPS was mainly located on top of the biomass and

438

contained only a few single cells, showing rather similarity with transparant exopolymers

439

(TEP) in freshwater, wastewater and seawater which are mainly composed of

440

polysaccharides37. As EPS production occurs at all feeding conditions, it is not a particular

441

response to BAM neither to starvation. EPS can sequester organic carbon compounds

442

including pesticides and improve their availability for cells in the matrix12,31,37-39. BAM is

443

rather hydrophilic (KOW 100.7-101.54) and might interact with EPS of a polar nature such as

444

ConA stained polysaccharides38. However, the results with MSH1 mutant M1a100g deficient

445

in BAM degradation but still able to produce the EPS, suggested no sorption of BAM on

446

EPS.

447

BAM degradation at micropollutant concentrations. BAM degradation by MSH1 reached

448

around 85-90% removal at all BAM concentrations except for 1 µg/L BAM where 70%

449

removal was noted. The amidase BbdA converting BAM to 2,6-dichlorobenzoic acid

450

(DCBA) is continuously expressed and theoretically, the presence of MSH1 cells assures the 17 ACS Paragon Plus Environment


Page 18 of 32

451

presence of BAM degrading enzymes irrespective of the BAM concentration22. BbdA has an

452

extreme low KM (135 µg/L or 0.7 µM) indicating high affinity for BAM22. Suspended MSH1

453

cells in batch showed a KS of 100±13 µg/L (0.5±0.1 µM) and a VMAX of 5.4±0.2 x 10-10 µg

454

BAM/cell/min (2.8±0.1 x 10-10 µmol BAM/cell/min). Since KM and KS are highly similar,

455

BAM degradation at low concentrations appears not limited by its uptake. Specific BAM

456

degradation rates in suspension were similar to those reported earlier for concentrations

457

ranging between 123 and 1000 µg/L, i.e., 10-10 µg BAM/cell/min39, 40.

458

However, sessile cells in the flow cells showed a KS of 630±58 µg/L (3.3±0.3 µM) and a

459

VMAX of 1.7±0.05 x 10-11 µg BAM/cell/min (9.0±0.3 x 10-14 µmol BAM/cell/min) (based on

460

biovolumes). Planktonic cells were not considered to calculate specific degradation rates

461

since they only composed a limited fraction in the channels (see above). The specific BAM

462

degradation rate at concentrations of 1 µg/L BAM and 1 mg/L of sessile cells (based on

463

biovolume) were respectively approx. 100 and 47-fold lower than those of suspended fresh

464

cells. Differences between specific degradation rates of suspended and sessile cells can be

465

due to substrate mass transfer limitation into and through the biofilm which depends on the

466

pollutant bulk concentration13. However, if diffusion is rate limiting than specific degradation

467

rates for the sessile cells will converge with those for suspended cells with increasing

468

concentrations which was not the case. Another reason could be differences in supply of

469

oxygen. However, oxygen levels in our study are close to saturation in the influent medium

470

and, since for BAM concentrations of 1 mg/L and lower, biofilms developed a thickness of

471

15 µm or lower, oxygen limitation is unlikely13. We hypothesize that instead cells in the flow

472

channels have a reduced activity or that only a fraction of the cells actively degrade BAM

473

despite the consitutive character of bbdA. In case of a feed of 1 µg/L this could be due to the

474

presumed VBNC state and reduced activity of a large fraction of cells as reported above due

475

to the long term starvation conditions. Similarly, Sjöholm et al.33 reported that with extended

476

period of starvation, a decreasing fraction of intact MSH1 cells contributed to mineralisation 18 ACS Paragon Plus Environment

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477

of BAM in suspended batch systems. Even viable cells (i.e., cells forming CFU) showed 10

478

to 20 times lower specific degradation activity compared to fresh cells in suspended batch

479

conditions (Table 2). MSH1 cells with reduced BAM degrading activity can be explained by

480

reduced BbdA production due to physiological responses to starvation such as the

481

intracellular degradation of proteins and/or RNA including BbdA and/or bbdA transcripts41.

482

Based on the BAM removal data of experiment 2, we calculated the specific BAM

483

degradation rate in flow systems receiving 1 µg/L BAM 1 day after inoculation, as 4.1±0.3 x

484

10-12 µg BAM/cell/min, i.e., similar to this of suspended fresh MSH1 cells in batch,

485

supporting our hypothesis that long term starvation conditions affect degradation. A similar

486

explanation can be assumed for 1 mg/L BAM which corresponds with 125 µg/L AOC close

487

to the average AOC found in groundwater and presumably as additional AOC in our flow

488

chambers (see above). As such 1 mg/L BAM can still invoke carbon starvation, although

489

apparently not to a level that leads to VBNC cells since the specific BAM degradation

490

ratebased on CFU at 1 mg/L BAM was similar to this based on biovolume estimates.

491

Otherwise, only the upper cells of the biofilm might have contributed to effective BAM

492

degradation while cells in deeper layers thrived on BAM degradation products. In contrast to

493

the observations with MSH1, specific degradation rates where highly similar in suspended

494

and flow systems for a linuron degrading Variovorax sp. strain at 10 µg/L, 100 µg/L and 1

495

mg/L linuron28 indicating that starvation effects might be organism depended.

496

Interestingly, predicted specific BAM degradation rates (based on biovolume) in the flow

497

systems at steady state were 10-100 times lower than the reported maximum specific

498

degradation rates in MSH1 inoculated sand columns receiving waters with 0.2 µg/L (4.0 x 10-

499

13

500

model deduced from the flow chamber experiments (Figure 3), specific degradation rates in

501

the flow channels should be 5.5 x 10-15 and 8. 2 x 10-14 µg/cell/min in case of a 0.2 µg/L and

502

3 µg/L BAM feed, respectively. The specific degradation rates for the sand column studies,

µg/cell/min). and 3 µg/L BAM (1.0 x 10-12 µg/cell/min)29, 42. Based on the kinetic Monod



Page 20 of 32

503

however, were calculated based on removal rates and MSH1 cell densities recorded during

504

the first 2 to 5 days following inoculation when cells are expected to not be yet starving33. As

505

reported above, at the start of the flow chamber experiment, specific degradation rates were

506

identical to those recorded for suspended systems.

507

In conclusion, our study provides deeper insight of the physiology and activity of pesticide

508

degrading bacteria in attached mode in continuous systems in case of micropollutant

509

concentrations, oligotrophy and starvation conditions (with focus on C and N deprevation)

510

and their application for removing micropollutants in biofilter units in DWTPs. Batch

511

systems using freshly grown cells can not always predict the cell activity under continuous

512

starvation conditions and the physiolgical state of the cell upon starvation, which might be

513

organism dependent, has to be considered. This way an alternative Monod model for

514

describing the kinetics of sessile MSH1 cells under N and C limited conditions was presented

515

but still needs to be validated in real systems. Evidence was provided that other AOC and/or

516

N controls the survival/growth of MSH1 and its BAM biodegradation/assimilation activity at

517

trace concentrations in DWTP biofilter units. Competition for the surplus AOC and/or N with

518

resident microbes in biofilters might further affect MSH1 colonisation and physiological

519

state43 as well as possible effects of phosphate limitation44. Ongoing research focusses on the

520

mechanism of the reduction of BAM degrading activity in strain MSH1 upon starvation and

521

the effect of indigeneous organisms on MSH1 colonisation.

522 523

Supporting Information Available

524

Includes information on Determination of BAM concentrations by UHPLC-MS/MS;

525

Determination of relative biovolume values in flow channels; Calculation of kinetic

526

parameters of BAM degradation in suspended batch cultures and flow chambers;

527

Determination of MSH1 Assimilable organic carbon (AOC) in the medium percolating the 20 ACS Paragon Plus Environment

Page 21 of 32


528

flow chambers; Tables Table S1. Total biomass production and yield estimation of MSH1-

529

GFP/M1a100g in experiments 2 and 3 for different feed concentrations of BAM; Figures

530

Figure S1: Effective influent BAM concentrations and effluent BAM concentrations in

531

abiotic controls recorded in the flow chamber experiments 1, 2 and 3; Figure S2A: CLSM

532

images from flow chambers inoculated with MSH1 and fed with 100 mg/L, 60 mg/L, 10

533

mg/L and 1 mg/L BAM in experiment 1; Figure S2B: CLSM images from flow chambers

534

inoculated with MSH1 and fed with 100 µg/L, 10 µg/L and 1 µg/L BAM and abiotic control

535

fed with 1 µg/L BAM in experiment 1; Figure S3: Biovolumes of MSH1 at front (black),

536

middle (dark gray) and back (light gray) section of the flow chamber as recorded at the end of

537

experiment 1; Figure S4: BAM degradation in flow chambers inoculated with MSH1-GFP

538

fed with either (A) 1µg/L BAM and (B) 1 mg/L BAM in experiment 2; Figure S5: Fraction of

539

biovolume of GFP expressing biomass of the total biovolume (being the combined GFP

540

expressing and Syto62 stained biovolume) in flow channels fed with no BAM and nominal

541

concentrations of 1 µg/L BAM and 1 mg/L BAM in experiment 2; Figure S6: Recorded EPS

542

per MSH1-GFP/M1a100g cell in flow chambers in experiment 3 fed with 1 mg/L, 1 µg/L and

543

no BAM; Figure S7: BAM degradation in flow chambers inoculated with either strain

544

MSH1-GFP or mutant M1a100g defective in BAM degradation and fed with either 1µg/L

545

BAM or 1 mg/L BAM in experiment 3. This material is available free of charge via the

546

Internet at http://pubs.acs.org.

547 548

Author contribution

549

Experimental design and writing of the manuscript was done by A.S, B.H and D.S. The main

550

body of laboratory experiments was performed by A.S and B.H. L.v.H. and J.Vd.B

551

performed the UHPLC-MS/MS analyses. J.H. was involved in the CLSM nalaysis. J.A. and

552

S.R.S. provided strain MSH1 and corresponding valuable information as well as being

553

involved in discussion writing. 21 ACS Paragon Plus Environment


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554

Acknowledgments

555

This study was funded by FP7 projects BIOTREAT (EU grant n° 266039) and

556

GOODWATER (EU grant n° 212683), the Inter-University Attraction Pole (IUAP) “µ-

557

manager” of the Belgian Science Policy (BELSPO, P7/25), the Danish Strategic Research

558

Council (Miresowa Project, grant no. 2104-08-0012), the FWO post-doctoral fellow grant n°

559

12Q0215N to B.H. and the C14/15/043 project of KU Leuven. We thank D Grauwels and K.

560

Simoens for technical assistance.

561

References

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646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695

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29. Albers, C. N.; Feld, L.; Ellegaard-Jensen, L.; Aamand, J., Degradation of trace concentrations of the persistent groundwater pollutant 2,6-dichlorobenzamide (BAM) in bioaugmented rapid sand filters. Water Res. 2015, 83, 61-70. 30. Polanska, M.; Huysman, K.; van Keer, C., Investigation of assimilable organic carbon (AOC) in flemish drinking water. Water Res. 2005, 39, (11), 2259-2266. 31. Tarao, M.; Seto, M., Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism. Appl. Environ. Microbiol. 2000, 66, (2), 566-570. 32. Helbling, D. E.; Hammes, F.; Egli, T.; Kohler, H.-P. E., Kinetics and yields of pesticide biodegradation at low substrate concentrations and under conditions restricting assimilable organic carbon. Appl. Environ. Microbiol. 2014, 80, (4), 1306-1313. 33. Sjøholm, O. R.; Nybroe, O.; Aamand, J.; Sørensen, J., 2,6-Dichlorobenzamide (BAM) herbicide mineralisation by Aminobacter sp. MSH1 during starvation depends on a subpopulation of intact cells maintaining vital membrane functions. Environ. Pollut. 2010, 158, (12), 3618-3625. 34. Pinto, D.; Santos, M. A.; Chambel, L., Thirty years of viable but nonculturable state research: Unsolved molecular mechanisms. Crit. Rev. Microbiol. 2015, 41, (1), 61-76. 35. Hunt, S. M.; Werner, E. M.; Huang, B.; Hamilton, M. A.; Stewart, P. S., Hypothesis for the role of nutrient starvation in biofilm detachment. Appl. Environ. Microbiol. 2004, 70, (12), 7418-7425. 36. Gjermansen, M.; Ragas, P.; Sternberg, C.; Molin, S.; Tolker-Nielsen, T., Characterization of starvation-induced dispersion in Pseudomonas putida biofilms. Environ. Microbiol. 2005, 7, (6), 894-904. 37. Berman, T.; Parparova, R., Visualization of transparent exopolymer particles (TEP) in various source waters. Desalination and Water Treatment 2010, 21, (1-3), 382-389. 38. Strathmann, M.; Wingender, J.; Flemming, H.-C., Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 2002, 50, (3), 237-248. 39. Simonsen, A.; Badawi, N.; Anskjær, G.; Albers, C.; Sørensen, S.; Sørensen, J.; Aamand, J., Intermediate accumulation of metabolites results in a bottleneck for mineralisation of the herbicide metabolite 2,6-dichlorobenzamide (BAM) by Aminobacter spp. Appl. Microbiol. Biotechnol. 2012, 94, (1), 237-245. 40. Schultz-Jensen, N.; Knudsen, B. E.; Frkova, Z.; Aamand, J.; Johansen, T.; Thykaer, J.; Sørensen, S., Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1. Appl. Microbiol. Biotechnol. 2013, 98, (5), 10. 41. Fan, C. L.; Rodwell, V. W., Physiological consequences of starvation in Pseudomonas putida: degradation of intracellular protein and loss of activity of the inducible enzymes of L-arginine catabolism. J. Bacteriol. 1975, 124, (3), 1302-1311. 42. Albers, C. N.; Jacobsen, O. S.; Aamand, J., Using 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in flow through biofilters—initial adhesion and BAM degradation potentials. Appl. Microbiol. Biotechnol. 2013, 98, (2), 957-967. 43. Liu, L.; Helbling, D. E.; Kohler, H.-P. E.; Smets, B. F., A model framework to describe growth-linked biodegradation of trace-level pollutants in the presence of coincidental carbon substrates and microbes. Environ. Sci. Technol. 2014, 48, (22), 1335813366. 44. Miettinen, I. T.; Vartiainen, T.; Martikainen, P. J., Phosphorus and bacterial growth in drinking water. Appl. Environ. Microbiol. 1997, 63, (8), 3242-5.


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696 697 698


Table 1. Overview of the BAM feed concentrations, operation times, MSH1/MSH1-GFP/M1a100g biomass in the flow channels, and BAMdegrading activity in experiments 1, 2 and 3. BAM feed concentration at end of experimentI Nominal

Used MSH1 variant

Operation time

Degradation extentII at steady state

Degradation rateIII at steady state

Actual Days

%

Cell numbers at steady state

Specific degradation rate at steady state

BiovolumeIV

CFUV

BiovolumeVI

CFUVII

Cells

Cells

µg BAM/ cell/day

µg BAM/ cell/day

2±1 x 1010 A* 3±1 x 1010 A* 2±1 x 1010 A* 2±1 x 109 B* 1±0 x 109 C* 9±1 x 108 C* 10±2 x 108 C* 6±1 x 108 D*

5±2 x 1011 A** 2±2 x 1011 A* 5±1 x 1010 B* 2±2 x 109 C* 3±2 x 108 D** 2±0 x 108 D** 1±0 x 108 D** 1±0 x 108 D**

3±0 x 10-7 A 1±0 x 10-7 B 5±0 x 10-8 C 5±0 x 10-8 D 6±0 x 10-9 E 9±0 x 10-10 F 6±0 x 10-11 G NA

2±0 x 10-8 A 2±0 x 10-8 B 2±0 x 10-8 C 5±0 x 10-8 D 2±0 x 10-8 C 5±0 x 10-9 5±0 x 10-10 F NA

2±0 x 109 A * 8±2 x 108 B * 7±1 x 108 B *

2±1 x 109 A * 3±1 x 108 B ** 4±3 x 108 B *

5±1 x 10-8 A 2±0 x 10-10 B NA

5±1 x 10- 8 A 4±0 x 10-10 B NA

2±0 x 109 A * 7±2 x 108 B * 5±0 x 108 B *

1±1 x 109 A * 6±4 x 106 B ** 4±2 x 106 B **

7±0 x 10- 8 A 2±0 x 10-10 C NA

10±0 x 10- 8 A 2±0 x 10- 8 C NA

2±1 x 108 D * 3±1 x 108 D * 1±0 x 108 E *

6±2 x 106 8±4 x 106 2±1 x 106

NA NA NA

NA NA NA

µg BAM/day A

100 mg/L 60 mg/L 10 mg/L 1 mg/L 100 µg/L 10 µg/L 1 µg/L No BAM

102 mg/L 59mg/L 14 mg/L 1.4 mg/L 93 µg/L 12 µg/L 1 µg/L No BAM

MSH1 MSH1 MSH1 MSH1 MSH1 MSH1 MSH1 MSH1

56 62 62 53 51 51 51 51

94±3 82±1 C 95±0 A 90±2 B 88±4 B 89±2 B 70±2 D NA

1 mg/L 1 µg/L No BAM

1.5 mg/L 1.5 µg/L No BAM

MSH1-GFP MSH1-GFP MSH1-GFP

48 28 28

91±11 A 84±3 A NA


1.7 mg/L 2 µg/L No BAM

MSH1-GFP MSH1-GFP MSH1-GFP

28 28 28

86±2 A 86±3 A NA


1.7 mg/L 2 µg/L No BAM

M1a100g M1a100g M1a100g

28 28 28

NA NA NA

Experiment 1 7788±237 4058±286 1091±2 106±2 6.70±0.30 0.83±0.02 0.05±0.00 NA Experiment 2 108±13 0.13±0.00 NA Experiment 3 118±3 0.13±0.00 NA NA NA NA

B ** B ** B **

25

ACS Paragon Plus Environment


699 700 701 702 703 704 705 706 707 708 709 710 711 712 713

Page 26 of 32

In all cases, values are the average of 3 flow channel replicates (n=3) with indicated standard deviations. IActual concentrations are the concentrations measured in the feed and/or the outlet of the abiotic control (in case available) at the end of the experiment just before stopping the experiment (when steady state was assumed) and were used to calculate overall and specific degradation rates. IIPercentage of BAM removed from influent at the end of the experiments representing steady state conditions. IIIRate of removal of BAM at the end of the experiments calculated as the difference between inlet and outlet BAM concentration (µg/L) multiplied by the flow rate of 0.082 L/day. IVMSH1/MSH1GFP/M1a100g cell numbers in the flow channel at the end of the experiments as estimated from their total biovolume using COMSTAT and taking into account the MSH1 cell volume. VMSH1/MSH1-GFP/M1a100g cell numbers in the flow channels at the end of the experiment as estimated by CFU counting after plating of cells extracted from the flow channels; VISpecific rates of degradation of BAM at the end of the experiment based on MSH1 biovolume in the flow channels; VIISpecific rates of degradation of BAM at the end of the experiment based on MSH1 CFU numbers the flow channels; NA: not applicable. Different capital letters (A-E) in superscript next to the presented values indicate significancy differences between values obtained for different feeding conditions within the same experiment as analyzed by one way ANOVA at a confidence level of 0.05. Before treating the values for ANOVA they were transformed to normal distribution. Different asterisks next to the values indicate significancy differences between values of MSH1/MSH1-GFP/M1a100g cell numbers counted by CFU or based on cell biovolume obtained for the same feeding condition in the same experiment as analyzed by the unpaired two tailed Student’s t-test (significance level 0.05).

26


Page 27 of 32

714 715 716 717


Table 2: BAM degradation rates and specific degradation rates in suspended batch cultures inoculated with freshly grown MSH1-GFP cells and comparison of specific degradation rates in suspension with those in flow chambers (at end of experiment when steady state was assumed). Standard deviations (n=3) are indicated. Initial BAM concentrationI

Degradation rate Suspended cells

µg/L 1±0 5±0 10±0 47±2 102±1 501±13 911±3

718 719 720 721 722 723 724 725

I II

III

Specific degradation rate Suspended cells

µg BAM/L/min 0.004±0.000 0.018±0.000 0.035±0.001 0.137±0.006 0.241±0.024 0.495±0.041 4.709±0.055

Flow chamber (biovolume)II

Flow chamber (CFU)III

µg BAM/cell/ min -12

4±0 x 10 2±0 x 10-11 4±0 x 10-11 1±0 x 10-10 2±0 x 10-10 5±0 x 10-10 5±0 x 10-10

3±0 x 10-14 1±0 x 10-13 3±0 x 10-13 1±0x 10-12 2±0 x 10-12 8±0 x 10-12 1±0 x 10-11

4±0 x 10-13 2±0 x 10-12 3±0 x 10-12 9±0 x 10-12 1±0 x 10-11 2±0 x 10-11 2±0 x 10-11

Initial BAM concentration in the suspended batch prior to inoculation. Calculated with the Monod kinetic model (v = vmax * [S/(S+KS)] with vMAX = 1.7±0.1 x 10-11 µg/cell/min and KS = 629±58 µg/L) based on data from flow channel experiment 1. The used numbers of MSH1 cells in flow channels were based on MSH1 biovolume estimates (R² = 0.98) Calculated with the Monod kinetic model (v = vmax * [S/(S+KS)] with vMAX = 1.7±0.2 x 10-11 µg/cell/min and KS = 42±28 µg/L) based on data from flow channel experiment 1. The used numbers of MSH1 cells in flow channels were based on MSH1 CFU estimates (R² = 0.95)

27



726

Page 28 of 32

Figure legends

727 728

Figure 1. Residual BAM concentrations (%) in function of time in the effluents of flow

729

channels inoculated with strain MSH1 and fed with (A) macropollutant nominal

730

concentrations of BAM: 100 (●), 60 (▲), 10 (■) and 1 (♦) mg/L BAM and (B)

731

micropollutant nominal concentrations of BAM: 100 (○), 10 (□) and 1 (◊) µg/L BAM in

732

experiment 1. Values of residual BAM % in the effluent of the inoculated channels fed with

733

BAM macroconcentrations are for each time point calculated based on the measured influent

734

concentration (see SI Figure S1). Values of residual BAM % in the effluents of the inoculated

735

channels fed with BAM microconcentrations are for each time point calculated in relation to

736

the value in the corresponding abiotic control (set as 100% - see SI Figure S1). Values are

737

averages with standard deviation represented by the error bars (n = 3)

738 739

Figure 2. MSH1-GFP biofilms recorded with CLSM at the end of the experiment in flow

740

channels fed with 1 mg/L BAM, 1 µg/L BAM and no BAM in experiment 2. (A) Top view of

741

the CLSM micrographs with shadow projection and cross sections in the x-z and y-z plane.

742

Biomass of MSH1-GFP was visualized by recording GFP production (green) and counter

743

staining with SYTO® 62 (red). (B) Sequential visualization of MSH-GFP biomass in flow

744

channels fed with 1 µg/L BAM: GFP (green) visualization (left); GFP (green) and SYTO® 62

745

(red) visualisation (middle); GFP (green), SYTO® 62 (red) and conA-TRITC (purple) (EPS)

746

visualisation (right). (C) Top view with shadow projection and cross sections in the x-z and

747

y-z plane of CLSM images of MSH1-GFP biomass in flow channels fed with 1mg/L (left),

748

1ug/L (middle) and no BAM (right). Biomass of MSH1-GFP was visualized by recording

749

GFP production (green) and counter staining with SYTO® 62 (red). EPS (purple) was

750

visualized with conA-TRITC. In all cases, shown images are those recorded at the front

751

section of the flow channel.

752 753

Figure 3: Aminobacter sp. MSH1-GFP/MSH1 BAM specific degradation rates in suspension

754

in batch using freshly grown cells (■) and in flow channels based either on biovolume (▲) or

755

CFU (●) in function of the initial BAM concentration. Experimental data were fitted by the

756

Monod kinetic model (dashed line).


Page 29 of 32


Abstract Graphic 196x164mm (300 x 300 DPI)



Figure 1. Residual BAM concentrations (%) in function of time in the effluents of flow channels inoculated with strain MSH1 and fed with (A) macropollutant nominal concentrations of BAM: 100 (●), 60 (▲), 10 (■) and 1 (♦) mg/L BAM and (B) micropollutant nominal concentrations of BAM: 100 (○), 10 (□) and 1 (◊) µg/L BAM in experiment 1. Values of residual BAM % in the effluent of the inoculated channels fed with BAM macroconcentrations are for each time point calculated based on the measured influent concentration (see SI Figure S1). Values of residual BAM % in the effluents of the inoculated channels fed with BAM microconcentrations are for each time point calculated in relation to the value in the corresponding abiotic control (set as 100% - see SI Figure S1). Values are averages with standard deviation represented by the error bars (n = 3) Figure 1 296x209mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32


Figure 2. MSH1-GFP biofilms recorded with CLSM at the end of the experiment in flow channels fed with 1 mg/L BAM, 1 µg/L BAM and no BAM in experiment 2. (A) Top view of the CLSM micrographs with shadow projection and cross sections in the x-z and y-z plane. Biomass of MSH1-GFP was visualized by recording GFP production (green) and counter staining with SYTO® 62 (red). (B) Sequential visualization of MSH-GFP biomass in flow channels fed with 1 µg/L BAM: GFP (green) visualization (left); GFP (green) and SYTO® 62 (red) visualisation (middle); GFP (green), SYTO® 62 (red) and conA-TRITC (purple) (EPS) visualisation (right). (C) Top view with shadow projection and cross sections in the x-z and y-z plane of CLSM images of MSH1-GFP biomass in flow channels fed with 1mg/L (left), 1ug/L (middle) and no BAM (right). Biomass of MSH1-GFP was visualized by recording GFP production (green) and counter staining with SYTO® 62 (red). EPS (purple) was visualized with conA-TRITC. In all cases, shown images are those recorded at the front section of the flow channel. Figure 2 148x133mm (300 x 300 DPI)



Figure 3: Aminobacter sp. MSH1-GFP/MSH1 BAM specific degradation rates in suspension in batch using freshly grown cells (■) and in flow channels based either on biovolume (▲) or CFU (●) in function of the initial BAM concentration. Experimental data were fitted by the Monod kinetic model (dashed line). Figure 3 165x113mm (300 x 300 DPI)


Page 32 of 32

Surface Colonization and Activity of the 2,6-Dichlorobenzamide (BAM

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