Physiological Responses of Salinity-Stressed Vibrio sp. and the Effect

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Physiological Responses of Salinity-Stressed Vibrio sp. and the Effect on Biofilm Formation on Nanofiltration Membrane LanHee Kim, and Tzyy Haur Chong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02904 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Environmental Science & Technology

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Physiological Responses of Salinity-Stressed Vibrio sp. and the Effect

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on Biofilm Formation on Nanofiltration Membrane

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Lan Hee Kim,† Tzyy Haur Chong*,†,⊥

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†

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Nanyang Technological University, 1 Cleantech Loop, CleanTech One 06-08, Singapore 637141,

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Singapore

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⊥

Singapore Membrane Technology Centre, Nanyang Environment and Water Research Institute,

School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang

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Avenue, Singapore 639798, Singapore

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*Corresponding author (E-mail: [email protected], Tel: +65-6513-8126, Fax: +65-6791-0756)

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ACS Paragon Plus Environment


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ABSTRACT

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This study evaluated the effects of salinity on the physiological characteristics of Vibrio sp.

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B2 and biofilm formation on nanofiltration (NF) membrane coupons used in the high

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recovery seawater desalination process. The test conditions were at 0.6 M, 1.2 M, and 2.4 M

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sodium chloride (NaCl), equivalent to salinity of seawater, brine at 50% and 75% water

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recovery, respectively. High salinity inhibited the cell growth rate, but increased the viability

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and bacterial membrane integrity. In addition, protein and eDNA concentrations of salinity-

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stressed bacteria were increased at 1.2 M and 2.4 M NaCl. In particular, protein concentration

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was linearly correlated with the NaCl concentration. Similarly, less biofilm formation on the

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NF membrane coupon (without permeation flux) was observed by the salinity-stressed

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bacteria, however, the production of extracellular polymeric substances (EPS) was

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significantly increased as compared to control; and protein was an influential factor for

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biofilm formation. This study shows that salinity-stressed bacteria have a high potential to

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cause biofouling on membrane surface as the bacteria still maintain the cell activity and

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overproduce EPS. The potential of biofilm formation by the salinity-stressed bacteria has not

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been reported. Therefore, the findings are important to understand the mechanisms of

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membrane biofouling under high salinity environment.

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Keywords: Salinity-stressed bacteria, Vibrio sp., Biofilm, Nanofiltration membrane

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INTRODUCTION

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Reverse osmosis (RO) membrane desalination has become a promising technology to

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produce high quality drinking water from seawater. However, low process recovery, which is

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typically below 50%, has been considered as a major disadvantage of the technology. Low

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recovery means high volume of discharge of brine which can adversely affect the marine

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ecosystems and increase the desalination cost due to treatment of brine before disposal.1-3

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Thus, high recovery of seawater desalination process is desired and has been explored. For

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the zero-discharge of brine (i.e. 100% recovery), technologies that have been employed

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include membrane distillation (MD) process and thermal methods such as brine concentrators

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or crystallizers.4-6 In addition, in our previous work, an energy-efficient reverse osmosis

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(EERO) process, which combines conventional single-stage reverse osmosis (SSRO) with a

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countercurrent membrane cascade with recycle (CMCR), can achieve a significantly higher

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water recovery (i.e. 75%) at a reduced osmotic pressure differential relative to SSRO. The

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CMCR consists of countercurrent retentate and permeate flow, permeate recycle, and

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retentate self-recycling via one or more nanofiltration (NF) stages.7,

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recovery desalination process was limited in practice due to membrane fouling that can

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reduce the performance efficiency and increase the operational cost.6, 9 Implications of high

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recovery include increase in the concentrations of salts as well as potential foulants in the

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system, which may exacerbate the membrane fouling. Among the fouling types, biofouling

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has been considered a major problem to be solved. Bacteria are transported to the membrane

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surface due to permeation flux.

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membrane surface through electrostatic and hydrophobic interactions; bacteria are auto-

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aggregated and produce extracellular polymeric substances (EPS) that maintain the biofilm

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structure by providing mechanical stability and protect bacteria from biocides via preventing

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the mass transport of antibiotics through the biofilm.9, 10

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However, high

Biofouling is initiated by attachment of bacteria on

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Environmental stresses such as salt stress can cause physiological changes to the

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microorganisms and influence the subsequent biofilm formation. High external osmolarity

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may cause plasmolysis and dehydration in some bacteria that lead to the inhibition of

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physiological processes such as nutrient uptake and DNA replication.11, 12 To cope with the

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salinity stress environment, microorganisms actively responded to maintain turgor and cell

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volume by osmoadaptation reaction.12 First, two stages of osmoadaptation responses occurred

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consecutively: uptake of potassium (K+) and accumulation of osmoprotectants.13 As a rapid

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response of osmotic up-shock, K+ uptake was performed by K+ uptake transport systems to

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balance the net negative charge of cytoplasmic macromolecules. When the salt concentration

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was above certain threshold level, a secondary response, the accumulation of osmoprotectants

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such as glycine betaine, carnitine and proline, was triggered.13 Second, the production of EPS,

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in terms of carbohydrate and protein, was enhanced as a protective response of bacteria under

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salinity stress.14-17 The pool of compatible solutes in salinity-stressed bacteria was finely

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tuned by uptake, synthesis, and excretion.12 The excretion of compatible solutes resulted in

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the enhancement of EPS content and the decrease of intracellular compatible solutes

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accumulation.17 It has been reported that EPS helped the bacteria to survive sodium toxicity

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by acting as a diffusion barrier.18 Last, expression of osmoregulated promoters, such as

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different σ-factors, enhanced the polysaccharide production and contributed to biofilm

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formation on the surface.12 For instance, the overexpression of σB factor in halo-tolerant

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Gram-positive bacteria such as Staphylococcus sp. and Bacillus subtilis were stimulated to

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form biofilm in microtitre plate assay under high salt concentrations by increased production

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of polysaccharide intercellular adhesion.19

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To the best of our knowledge, however, the study on the influence of high salinity condition 4


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on the physiological characteristics of bacteria and biofilm formation on the membrane

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surface in seawater desalination is limited. For instance20, various feed salinities of low (38

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g/L – 40 g/L TDS), medium (41 g/L – 44 g/L TDS) and high (46 g/L – 50 g/L TDS), or

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equivalent to 1.1 – 1.4 × seawater salinities, prepared by blending of sand-filtered seawater

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and concentrate from a full scale seawater desalination plant, were found to greatly affect the

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biofouling dynamics in terms of bacteria multiplication and EPS release in a crossflow RO

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membrane filtration unit. In general, ~ 85% decline in flux (from 1.3 to 0.2 m3/m2/d) and ~

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68% decline in rejection (from 95 to 30%) at constant pressure operation were observed at

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low salinity; this observation was correlated to higher reproduction of bacteria and greater

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EPS concentration. Meanwhile at medium and high salinities, marginal decline in the

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membrane performances (flux decreased from 0.64 to 0.52 and 0.54 to 0.42 m3/m2/d, and

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rejection decreased from ~ 95 to 80%, respectively) were observed though the total

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accumulated cells peaked at 105 cells/cm2 similar to low salinity condition. However, the

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impact of concentration polarization on the biofouling in RO process was not considered.

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Concentration polarization (CP) is a phenomenon arisen due to the accumulation of rejected

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solutes on the surface of RO membrane that causes the concentration at the membrane

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surface (Cw) higher than the bulk solution (Cb), in which its magnitude is an exponential

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function of the ratio of flux (J) ÷ mass transfer (k) i.e. Cw/Cb ~ exp (J/k). In the above study19,

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the initial flux at constant pressure and fixed crossflow operation at low feed salinity was

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greater (~ 2 ×) than at medium and high feed salinities, indeed as a result the actual salinity

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level was greater at the membrane surface where the biofilm was formed. Hence, the impact

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of salinity level on biofouling was not justified. More importantly, the nutrient level at the

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membrane surface, which was a limiting factor for biofilm formation, was greater under the

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CP effect at the higher flux condition to support greater production of bacteria cells and EPS.

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The impact of flux and nutrient levels on biofouling in RO has been well studied and reported 5



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in the literatures.21-25

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The microenvironment above the membrane surface is significantly influenced by multi-

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factors such as flux, cross flow, recovery, membrane rejection etc., which vary spatially from

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the first to the last element in an actual RO system26, 27. The condition is further complicated

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by the interplay between these parameters and fouling i.e. fouling is flux-driven, but on the

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other hand, fouling can reduce flux.28 Therefore, this study as an initial attempt to investigate

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the biofouling event in a high recovery desalination process focused on the physiological

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responses of salinity-stressed bacteria and the effects on biofilm formation on the membrane

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surface under static condition, i.e. no permeation flux and crossflow, to isolate the salinity

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factor and simplify the conditions encountered in an actual desalination system. To simulate

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the salinity levels of seawater, brine at 50% and 75% water recovery, 0.6 M, 1.2 M, and 2.4

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M NaCl, respectively, were selected. First, the physiological characteristics of salinity-

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stressed halo-tolerant bacteria Vibrio sp. B2 which was selected as a model bacterium in this

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study with high potential of biofilm formation under various NaCl concentrations ranging

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from 0.3 M (control) to 2.4 M were investigated. Second, the potential of biofilm formation

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by salinity-stressed bacteria on the NF membrane coupon was evaluated under no membrane

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permeation flux condition. NF membrane was used in this study to align with our previous

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work on EERO to achieve high recovery desalination process. This study can help to

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understand the mechanisms of biofilm formation by salinity-stressed bacteria and to propose

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strategies to combat biofouling in a membrane system for efficient water recovery in the

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desalination process.

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MATERIALS AND METHODS

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Selection of model bacterium. Bacteria were first isolated from seawater, brine, and

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biofouled membrane coupon. 5 mL of raw seawater and brine samples, collected from a

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seawater desalination plant in Singapore, were inoculated in 5 mL of marine broth 2216

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including peptone and yeast extract as organic nutrient sources (Table S1; Difco, USA) and

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incubated for 24 h at 37 ºC with continuous shaking. Biofouled membrane coupon, which

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was obtained by operating a lab-scale reverse osmosis (RO) membrane desalination system

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with raw seawater, was soaked in 10 mL of marine broth and incubated for 24 h at 37 ºC. All

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the enriched cultures from different sources were spread on the marine agar (BD, NJ, USA)

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and different forms of colonies on marine agar plate were incubated in a fresh marine broth

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for 24 h at 37 ºC with continuous shaking. To assess the potential of biofilm formation of

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isolated bacteria, microtiter plate biofilm assay was performed according to the methods as

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previously described.29 Briefly, the cultured cells were centrifuged at 4,234 × g for 20 min

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and washed with a phosphate buffered saline (PBS; Life Technologies, CA, USA) two times

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followed by centrifugation at 4,234 × g for 10 min. The cultures (optical density at 600 nm to

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1.0; OD600, 1.0) were incubated in a 96 well microplate (TPP, Trasadingen, Switzerland)

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containing marine broth at 37 ºC for 24 h. The microplate was washed with MiliQ water and

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stained with 0.1% crystal violet solution for 10 min at room temperature (RT). 250 µL of 95%

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ethanol was added to the dried microplate and incubated for 10 min. The OD600 of crystal

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violet-stained biofilm was measured by using a microplate reader (BioTek Synergy H1

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Hybrid Reader, VT, USA). From the microplate biofilm assay, bacteria were divided into 4

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categories: non-adherent, weakly adherent, moderately adherent, and strongly adherent

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bacteria and the cut-off OD (ODc) indicates the mean OD of the negative control (without

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cell inoculation).30 To further isolate the bacteria to be chosen as the model bacterium for

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subsequent studies, membrane biofilm assay was carried out. The NF membrane (NE4040-90;

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Toray Chemical Korea Inc., Seoul, Korea) was used in this study. It has −16 mV surface 7



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charge at pH 5.6 and 85-95% salt rejection under test condition with 2,000 mg/L of NaCl at 5

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bar (Table S2).31-33 The cultured cells (OD600, 1.0) were inoculated into a 6-well plate

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containing attached NE4040-90 NF membrane coupons (size of 2 × 2 cm2) and 5 mL marine

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broth. The plate was incubated at 37 ºC for 4 days to form biofilm on the NF membrane

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coupons. Bacteria having the highest potential of biofilm formation on the NF membrane

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coupon was selected as a model bacterium and identified by analysis of 16S rDNA sequence.

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Genomic DNA was extracted and polymerase chain reaction (PCR) was performed with

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universal

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GGTTACCTTGTTACGACTT-3′). The PCR reaction proceeded as follows: 95 ºC for 6 min,

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30 cycles of 94 ºC for 60s, 58 ºC for 60s, and 72 ºC for 90s, followed by an elongation step

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for 5 min at 72 ºC. The obtained DNA sequence was compared with database by doing the

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basic local alignment search tool (BLAST) search.

primers,

27F

(5′-AGAGTTTGATCATGGCTCAG-3′)

and

1492R

(5′-

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Effect of salt concentration on bacterial growth and viability. The OD600 of selected

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model bacterium, Vibrio sp. B2, was adjusted to 1.0 and 50 µL of cell culture was added into

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50 mL marine broth (contains background NaCl of 0.3 M) containing 0.6 M, 1.2 M, and 2.4

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M NaCl. Samples were taken at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 32 h, 48 h, and 60 h for

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measurement of OD600 and cell viability. To analyze cell viability, samples were stained with

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SYTO®9 and propidium iodide (PI) dyes (Molecular Probes, OR, USA) and cell numbers

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were measured by flow cytometer (BD, NJ, USA). The percentage of live cells was

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calculated by dividing the number of live cells by total cell numbers. In addition, the esterase

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activity was analyzed by using 10 µM carboxyfluorescein diacetate (cFDA; Life

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Technologies CA, USA) and PI staining dyes.34 For intracellular enzymatic conversion of

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cFDA to carboxyfluorescein, cFDA stained cells were incubated at 37 ºC for 30 min. PI was

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added to label the cells with damaged membrane in the cFDA stained cells and incubated in

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ice until analysis by flow cytometer (BD, NJ, USA).

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Characterization of extracellular polymeric substances (EPSs) and surface charge. EPS

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of salinity-stressed bacteria was extracted according to procedure in previous reference.35 In

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brief, 0.06 mL of formaldehyde (36.5%; Sigma-Aldrich, MO, USA) was added to 10 mL of

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sample and stored at 4 ºC for 1 h to prevent cell lysis by bacteria fixation. After cell fixation,

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4 mL of 1 N NaOH was added and incubated for 3 h at 4 ºC. Then sample was centrifuged at

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20,000 × g for 20 min and the supernatant was filtered through a 0.2 µm membrane and

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dialyzed against MiliQ water using a 3,500 Da dialysis membrane (Pierce, IL, USA) for 24 h.

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Lyophilized sample was re-suspended in 10 mL of MiliQ water. Carbohydrate (CH) content

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was determined by phenol-sulfuric acid method.36 Briefly, 600 µL of concentrated sulfuric

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acid was added to 200 µL of sample and 120 µL of 5% (w/v) phenol was added immediately,

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followed by incubation for 5 min at 90 ºC. The solution was transferred to a 96-well

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microplate and OD490 was measured by microplate reader (BioTek Synergy H1 Hybrid

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Reader, VT, USA). Glucose standard solution was used as calibration to calculate the

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carbohydrate concentration. Protein (PN) content was determined by using BCA assay kit

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(Thermo Scientific Inc., NH, USA) according to the manufacture’s guidelines. The

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concentration of extracellular DNA (eDNA) in EPS was determined by using Quant-iT

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PicoGreen® dsDNA kit (Molecular Probes, OR, USA) and the fluorescence intensity was

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analyzed by microplate reader (BioTek Synergy H1 Hybrid Reader, VT, USA). The three-

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dimensional excitation-emission matrix (EEM) of EPS was analyzed by fluorescence

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spectrophotometer (Perkin Elmer, MA, USA) under excitation of 220 nm to 450 nm and

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emission of 280 nm to 550 nm. The zeta potential of salinity-stressed bacteria was analyzed

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by zetasizer (Malvern Zetasizer nano-zs, Worcestershire, UK). 9



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Effect of abrupt changes in salt concentrations on bacterial growth and viability. The

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cultured bacteria were first inoculated in the marine broth containing 0.6 M NaCl and

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incubated for 26 h until stationary phase. After 26 h of incubation, bacterial cells were

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harvested by centrifugation at 4,234 × g for 20 min. Subsequently, the harvested cells were

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inoculated into different marine broths containing 0.6 M, 1.2 M, and 2.4 M NaCl and

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incubated for 24 h. Bacterial growth (OD600), viability, and esterase activity were measured.

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Metabolic activities of salinity-stressed Vibro sp. B2. The model bacterium was incubated

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for 16 h to reach the log phase. The respiratory activity was analyzed by using a BacLight

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RedoxSensor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) vitality kit (Molecular Probes,

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OR, USA) according to the protocol provided by manufacturer. To calculate total cell

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numbers, the sample was counter stained with SYTO®24 after staining with CTC reagent and

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analyzed by flow cytometer (BD, NJ, USA). The percentage of respiratory active cells was

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calculated via dividing the number of CTC positive cells by total cell numbers. The activity

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of DNA synthesis was analyzed by using a Click-iT® EdU flow cytometry assay kit

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(Molecular Probes, OR, USA). The ratio of EdU positive cells was calculated via dividing the

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number of Alexa Fluor® 488 azide stained cells by total cell numbers.

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Biofilm formation on NF membrane coupons (without membrane permeation flux).

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Vibrio sp. B2 was incubated in marine broth for 24 h and cultured cells were harvested and

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washed three times with PBS buffer. Cultured cells (OD600 1.0) were inoculated in a

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microplate including marine broth containing 0.6 M, 1.2 M, and 2.4 M NaCl and attached NF

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membrane coupons (2 × 2 cm2). To form biofilm on NF membrane, the microplates were

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incubated for 4 days under 90 rpm at 37 ºC. After 4 days, membrane coupons were washed 10


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three times with PBS buffer and the total cell number, cell viability and EPS concentrations

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were measured. The morphology and infrared fingerprint of biofilm formed on the NF

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membrane coupons were characterized by confocal laser scanning microscopy (CLSM; Carl

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Zeiss, Oberkochen, Germany) and attenuated total reflectance Fourier transform infrared

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spectroscopy (ATR-FTIR; Shimazu, Tokyo, Japan), respectively. In the CLSM analysis, to

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stain the attached biofilm, fouled membrane coupons were soaked in 1 mL PBS containing

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1µL each of SYTO®9 and PI dye, then washed with PBS three times and observed under

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CLSM equipped with an argon laser having excitation wavelengths of 458 nm, 488 nm, 514

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nm and 63 × plan-Apochromat objective. The images were captured by ZEN software

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(version 2.1, Carl Zeiss, Oberkochen, Germany) and surface area (µm2) or biovolume (µm3)

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of biofilm were calculated by IMARIS software (Bitplane, Zurich, Switzerland). The

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Spearman’s rank correlation coefficient (Rs) between NaCl concentrations and EPS

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concentrations were analyzed.37

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RESULTS AND DISCUSSION

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Selection of model bacterium. Total of 16 strains were categorized into strongly adherent

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bacteria and 3 strains were moderately adherent bacteria (BF4, BF8, B4) from the microtiter

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plate assay (Figure 1a). Among the strongly adherent bacteria, B2, B5, B6, and S5 strains that

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showed significant biofilm formation were selected for subsequent biofilm assay on NF

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membrane coupon. Strain B2 showed the highest potential of biofilm formation on NF

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membrane (Figure 1b). B2, B5, and B6 were identified as Vibrio sp. (similarity 98-99%) and

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S5 was highly similar with Shewanella haliotis (similarity 100%). Proteobacteria phylum has

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been shown to be the most abundant bacterial group in seawater and fouled RO membrane.38,

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39

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saline-MBR with increase in salinity.40 As the belonging γ-proteobacteria class, Vibrio and

Among the Proteobacteria phylum, the abundance of γ-proteobacteria were elevated in

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Shewanella species are well known biofilm-forming halo-tolerant bacteria in seawater reverse

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osmosis system.41 Therefore, Vibrio sp. B2 was selected as a model bacterium in the

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subsequent studies.

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Changes in physiological characteristics of salinity-stressed bacteria. The physiological

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characteristics of salinity-stressed bacterium, Vibrio sp. B2 were summarized in Table 1. As

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compared to control, 0.1-log, 3.0-log, and 3.3-log reductions in total cell numbers were

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observed and half-maximal inhibitory concentration (IC50) was 0.8 M NaCl that means

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growth of Vibrio sp. B2 was inhibited by 50% at concentration of 0.8 M NaCl. However,

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interestingly, cell viability was increased by 96.4% and 96.1% and membrane integrity

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(esterase activity %) which provides the information about degree of membrane injury was

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increased by 46.6% and 47.2% at 1.2 M and 2.4 M NaCl compared to control, respectively.

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The effect of salinity on cell viability and membrane integrity were shown in Figure S1. As

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the NaCl concentration increased from 0.3 M to 6.0 M, cell viability (Figure S1a) and

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membrane integrity (Figure S1b) were increased by 96.9% and 52.6% after 24 h incubation,

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respectively. As an osmoprotective activity, bacteria adapt their intracellular osmolarity or

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increase their cell wall integrity to survive in a broad range of solute concentrations.42

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However, the abrupt changes in salt concentrations showed different result in the

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physiological responses of bacteria exposed to higher salinity for 24 h. When the cultured

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cells at 0.6 M NaCl were transferred to a fresh medium with the same concentration of 0.6 M

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NaCl, cell growth rate and 34.7% of viability were recovered in 3 h after the transfer (Figure

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2a, 2b). However, when trasnferred from 0.6 M to higher salinities of 1.2 M and 2.4 M NaCl,

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cell growth dropped significantly, which was an indication of cell death; and membrane

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integrity was decreased by 4.4% and 16.0%, respectively (Figure 2a, 2c). This was because

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bacteria could not adjust to the sudden changes of salinity within a short time. Bacterial cells 12


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abruptly exposed to the high salinity medium may stay in a stage of plasmolysis which

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resulted in the inhibition of physiological activities.11

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On the other hand, protein concentration was increased by 3.1-log and 2.4-log, eDNA

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concentration was increased by 3.3-log and 2.4-log at 1.2 M and 2.4 M NaCl compared to

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control, respectively. However, the carbohydrate concentration was not affected by the

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increase in salinity level. The increase in protein and eDNA at 1.2 M and 2.4 M NaCl caused

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a strong negative bacteria surface charge (Table 1). The concentration of protein was

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significantly correlated with the concentration of NaCl (Rs = 1.0, p < 0.05), while the

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correlation of carbohydrate concentration with NaCl concentration was not considered as

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statistically significant (Rs = 0.8, p > 0.05). Increase in EPS production under high salt

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condition can be caused by secretion of osmoprotectants which are accumulated water-

311

soluble, low-molecular-weight intracellular organic compounds or autolysis of cells.16, 43, 44

312

The EEM fluorescence spectra help to understand the changes of EPS compositions in terms

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of humic-like, protein-like, fulvic acid-like substances and tyrosine-like proteins under

314

various salinity conditions. The EEM plot also showed that intensities of protein-like

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substance (II; Ex = 250-280 nm, Em < 380 nm) and tyrosine-like substances (IV; Ex = 220-

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250 nm, Em = 330-380 nm)45 were increased by 25.1%, 14.7%, 24.4%, and 5.9%, 8.0%,

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16.4%, respectively at 0.6 M, 1.2 M, and 2.4 M NaCl compared to control, respectively.

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However, peak intensities of humic-like substance (I; Ex > 280 nm, Em > 380 nm) and fulvic

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acid-like substance (III; Ex = 220-250 nm, Em > 380 nm)45 were reduced by 36.8%, 34.3%,

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and 17.4%, 9.6% at 1.2 M and 2.4 M NaCl compared to control, respectively (Figure 3). The

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increase in peak intensity of humic-like substance at 0.6 M NaCl compared to control

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probably caused by the decomposition of dead cells and macromolecular organics such as

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proteins and polysaccharides.46

324 13



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Metabolic activities of salinity-stressed bacteria. Respiratory activity were increased by

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8.3%, 85.5%, and 87.0% at 0.6 M, 1.2 M, and 2.4 M NaCl compared to control, respectively

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(Figure 4a). The CTC assay indicates the respiratory activity of bacterial cells. A colorless

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CTC staining dye is converted to red-fluorescent precipitate by the electron transport system

329

of respiratory-active bacterial cells.47 The increase of CTC-positive cells can be caused by the

330

development of cell population and the elevated respiratory-active state of cells.47 In our

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study, CTC-positive cell numbers were reduced, and a decrease in total number of bacteria

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cells under high salt concentrations were observed. However, the ratio between CTC-positive

333

cell number and total cell numbers were significantly increased at 1.2 M and 2.4 M NaCl

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concentrations. This means the salinity-stressed bacteria still maintain respiratory activity and

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the result was consistent with the increase in cell viability (Table 1). At 1.2 M and 2.4 M

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NaCl, bacterial activation was highly increased as compared to control and 0.6 M NaCl. This

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could be a result of the adaptation mechanism of bacteria to hyper-osmotic shock

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environment. Gram-negative bacteria counteract to high salinity by stimulation of potassium

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(K+) uptake and accumulation of osmoprotectant such as glycine betaine, proline.13 The K+

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uptake is performed via K+ transport systems associated with adenosine triphosphate (ATP)

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hydrolysis; therefore, elevated osmolarity can increase the respiratory activity to generate

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ATP to be used for K+ uptake. To measure the bacterial ability to proliferate, DNA synthesis

343

activity of salinity stressed bacteria was measured by flow cytometric detection of

344

incorporated EdU, which is a thymidine analogue, during DNA synthesis.48 Salinity-stressed

345

bacteria in log phase showed that the ability of DNA synthesis for bacteria proliferation was

346

inhibited by 58.9%, 82.9%, and 82.3% at 0.6 M, 1.2 M, and 2.4 M NaCl compared to control,

347

respectively. However, they still have activity to continue cell proliferation (Figure 4b).

348

14


Page 14 of 34

Page 15 of 34


349

Biofilm formation on NF membrane coupons. As compared to control, total cell numbers

350

of biofilm formed on NF membrane coupons were reduced by 0.2-log, 1.7-log, and 1.8-log at

351

0.6 M, 1.2 M, and 2.4 M NaCl, respectively (Figure 5a). However, the cell viability was

352

18.9%, 7.7%, 30.2%, and 39.4% at control (0.3 M), 0.6 M, 1.2 M, and 2.4 M NaCl,

353

respectively (Figure 5b). The results were highly consistent with the in-vitro assay which

354

showed the effect of salt concentration on bacteria growth and viability (Table 1). The

355

concentrations of carbohydrate and protein per total cell numbers were increased by more

356

than 96% at 1.2 M and 2.4 M NaCl compared to control, respectively; and the ratio between

357

protein and carbohydrate (PN/CH) was 2.4, 2.1, 2.4, and 2.6 at control, 0.6 M, 1.2 M, and 2.4

358

M NaCl, respectively. Thus, protein produced by salinity-stressed bacteria was the dominant

359

compounds in biofilm. The concentration of protein (ng/cells) that was released from the

360

bacterial cells were highly correlated with NaCl concentration (Rs=1, P < 0.05), however,

361

carbohydrate concentration did not show linear correlation with NaCl concentration (Rs=0.8,

362

p > 0.05). These results were similar to salinity-stressed bacteria in suspension. The FTIR

363

peaks of biofilm formed on NF membrane coupons also showed that the peak intensities of

364

control sample were entirely smaller than other conditions (Figure 6). In particular, among

365

the FTIR peaks, the specific peak that assigned as RNA backbone (970 cm-1; νC-C, νP-O-P),

366

proteins/carbohydrates/esters (1153/1172 cm-1; νsC-OH, νC-O), proteins (1568-1531 cm-

367

1

368

increased at high NaCl concentrations (Table 2). EEM peaks showed that fulvic acid (III) and

369

tyrosine-like proteins (IV) were dominant on the membrane surfaces (Figure S2). The ratio

370

between tyrosine-like proteins and humic acid indicated that aromatic protein-like substances

371

such as tyrosine-like proteins (IV) were more influential than protein-like substances (II) to

372

cause biofilm (Table 3). The biofilm coverage (%) was reduced by 11.7% and 60.9% at 1.2 M

373

and 2.4 M NaCl, while at 0.6 M NaCl concentration, the biofilm coverage was increased by

/1693-1627 cm-1; Amide I/Amide II), hydroxyl (3000-3400 cm-1; -OH) were significantly

15



374

14.9% compared to control, respectively. However, the biomass surface to bio-volume ratio

375

(µm2/µm3), calculated by dividing the summation of biomass surface (µm2) by biovolume

376

(µm3) were 3.5 (±0.7), 3.7 (±0.8), 3.4 (±0.5), and 3.3 (±0.5) at control, 0.6 M, 1.2 M, 2.4 M

377

NaCl, respectively (Table S3). The biomass surface to bio-volume ratio indicates the actual

378

fraction of biofilm exposed to the nutrient on the membrane surface. Initially bacteria

379

exposed to the nutrient and the biofilm is matured to adapt to the environment. Therefore, the

380

biomass surface to bio-volume ratio may indicate the adaptation of biofilm to the

381

environment.49 The results indicated that although less biofilm was formed on NF membrane

382

coupons, the surface to bio-volume ratio was not affected since salinity-stressed bacteria

383

adapted to the high salinity by maintaining its membrane integrity and metabolic activity.

384 385

This study investigated the effect of salinity on physiological characteristics of Vibrio sp. B2

386

and the possible impact on biofilm formation on NF membrane coupon. We highlight that

387

although the total cell numbers are lower in higher salinity environment, the bacteria maintain

388

cell integrity, remain active and can overproduce EPS which may have high potential to cause

389

severe biofilm formation in the high recovery desalination system. The results can be

390

explained by uptake K+ ion with the active respiratory activity and subsequent production of

391

osmoprotectants as osmoadaptation mechanisms of bacteria to osmotic stress.12, 13 However,

392

in an actual desalination system, parameters such as feed water compositions,50, 51 membrane

393

properties,52 and hydrodynamic conditions (i.e. flux and cross-flow velocity)26, 27 can greatly

394

contribute to the membrane fouling dynamics. It has been reported from membrane autopsies

395

study that organic and biofouling mainly occurred at the lead membrane element, while more

396

serious organic fouling, scaling, and still significant biofouling were detected at the

397

concentrated brine end of an conventional RO system.51 In further study, the evaluation of

398

membrane biofouling for high recovery desalination process under long term operation of 16


Page 16 of 34

Page 17 of 34


399

membrane system is required. In addition, based on the results from the above studies on

400

physiological responses of salinity-stressed bacteria, it is predicted that the biofilm structures

401

and compositions would vary along the membrane modules and stages in the high recovery

402

desalination process since the concentration of salts increases from the inlet to outlet as water

403

is recovered. Thus, the biofilm with high quantity of active bacteria cells embedded in matrix

404

of EPS is expected at the first stage (i.e. inlet). On the other hand, the biofilm at the last stage

405

(i.e. outlet) would have greater amount of EPS especially protein and eDNA but with lower

406

total number of cells. Hence, different fouling control or cleaning strategies need to be

407

adopted. It is proposed to use the concentrated brine, i.e. × 4 times the original concentration

408

at 75% recovery, as an agent for osmotic-shock to control membrane fouling in the first stage.

409

The effect of abrupt change in salinity level has shown to reduce the viability and cell

410

membrane integrity of bacteria. The direct osmosis backwash cleaning technique has been

411

proposed for RO fouling control in water reuse application by periodical injections of high

412

salinity solution made from industrial grade NaCl (136 kg/m3 NaCl) to the feed water which

413

then induce the reverse flow of RO permeate that can lift and sweep off foulants from the

414

membrane surface.53 However, this method is not suitable for the last stage, which is at high

415

salinity level. Here, enzyme based cleaning agent54 or biosurfactant45 that target at protein

416

removal is desired.

417 418

ACKNOWLEDGEMENTS

419

This research grant is supported by the Singapore National Research Foundation,

420

administered by PUB. The Singapore Membrane Technology Centre, Nanyang Environment

421

and Water Research Institute, Nanyang Technological University is supported by the

422

Economic Development Board of Singapore.

423 17



424

Supporting Information. The compositions of marine broth 2261 (Table S1), characteristics

425

of NF membrane (Table S2), characteristics of biofilm formed on NF membrane coupons

426

(Table S3), cell viability and esterase activity of salinity-stressed bacteria in various NaCl

427

concentrations ranging from 0.3 M to 6.0 M NaCl (Figure S1), EEM plot of EPS extracted

428

from biofilm formed on NF membrane coupons (Figure S2) are provided.

429 430

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different is the composition of the fouling layer of wastewater reuse and seawater 23



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Structure, properties and performance relationship. Desalination 2013, 326, 77-95.

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581

enzymatic treatment on the reduction of extracellular polymeric substances (EPS) from

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583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 24


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600 601

Figure 1. (a) Adherence of isolated bacteria on microtiter plate. Bacteria isolated from

602

biofouled membrane (BF), brine (B), and seawater (S). (b) Potential of selected bacteria to

603

form biofilm on NF membrane (n=3).

604 605

25



606 607

Figure 2. Effects of abrupt changes in salt concentrations on (a) bacterial growth (OD600), (b)

608

viability, and (c) bacterial membrane esterase activity (n=3).

26


Page 26 of 34

Page 27 of 34


609

610

Figure 3. Excitation-emission matrix (EEM) of EPS extracted from salinity-stressed bacteria

611

at (a) control (0.3 M), (b) 0.6 M, (c) 1.2 M, and (d) 2.4 M NaCl. I : humic-like matter; II,

612

protein-like matter; III, fulvic acid-like substances; IV, tyrosine like proteins (n=3).

613 614 615 616 617 618

27



619

620

Figure 4. Metabolic activities of salinity-stressed bacteria, (a) respiratory activity compared

621

to total cell numbers (b) proliferation activity (%) of bacteria at control (0.3 M), 0.6 M, 1.2 M,

622

and 2.4 M NaCl (n=3).

623

624

28


Page 28 of 34

Page 29 of 34


625 626

Figure 5. Biofilm-forming potential of salinity-stressed bacterium, Vibrio sp. B2 on NF

627

membrane coupons, (a) total attached cell numbers, (b) the ratio of live cells to total cell

628

numbers, (c) EPS concentrations (n=3).

29



629 630

Figure 6. ATR-FTIR spectra of biofilm-formed NF membrane coupons caused by salinity-

631

stressed bacteria at control (0.3 M), 0.6 M, 1.2 M, and 2.4 M NaCl (n=3).

632

633

634

635

636

637

638

30


Page 30 of 34

Page 31 of 34

639


Table 1. Effects of NaCl concentrations on physiological characteristics of Vibrio sp. B2 NaCl concentration (M) Parameters Control (0.3) Total cell number (cells/mL)

640

7

8.3 × 10

5

0.6

1.2 7

8.1 × 10

6

2.4 4

8.3 × 10

4

4.6 × 10

4 3

(±7.1 × 10 )

(±3.5 × 10 )

(±3.3 × 10 )

(±6.1 × 10 )

Maximum specific growth rate (h-1)

0.086

0.062

0.013

0.009

Cell viability (Live %)

2.9 (±0.8)

2.0 (±0.7)

78.7 (±1.6)

74.1 (±3.8)

Esterase activity (cFDA %)

41.8 (±3.3)

50.7 (±6.7)

78.4 (±2.4)

79.2 (±1.0)

Carbohydrate (ng/cells)

1.6 × 10-4 (±5.8 × 10-6)

1.5 × 10-4 (±3.1 × 10-5)

1.8 × 10-4 (±6.8 × 10-6)

2.2 × 10-4 (±1.9 × 10-5)

Protein (ng/cells)

1.7 × 10-3 (±1.1 × 10-5)

2.0 × 10-3 (±3.0 × 10-4)

2.0 (±9.2 × 10-2)

3.6 (±0.3)

eDNA (ng/cells)

9.3 × 10-4 (±3.4 × 10-4)

9.4 × 10-4 (±4.2 × 10-4)

2.4 × 10-1 (±6.3× 10-2)

2.1 × 10-1 (±9.0 × 10-2)

Zeta potential (mV)a

-8.5 (±0.2)

-8.1 (±0.6)

-18.5 (±0.6)

-18.6 (±0.2)

a

pH 7, 25 °C (n=9)

641

31



Page 32 of 34

Table 2. Assignments of FTIR spectra of biofouled membranes which was formed by salinity-stressed bacteria Wavenumber (cm-1)

Assignment

970

νC-C, νP-O-P

RNA backbone

νsC-OH, νC-O

Proteins, carbohydrates, esters

1153 1172 1568-1531 1693-1627 3000-3400

Amide II (δN-H coupled with νC-N) Amide I (νC=O coupled with δN-H), δH2O Hydroxyl (-OH)

Principal compounds and/or functions

Absorbance Control (0.3 M)

0.6 M

1.2 M

2.4 M

0.077

0.144

0.130

0.176

-0.038

0.018

0.118

0.164

-0.014

0.048

0.106

0.150

Proteins

0.092

0.169

0.162

0.208

Proteins, water

0.092

0.165

0.162

0.209

-

0.079

0.155

0.174

0.223

32


Page 33 of 34


Table 3. EEM peak intensities of extracted EPS from salinity-stressed bacteria or biofilms

642

NaCl concentration (M) Control (0.3)

643

a

Salinity-stressed bacterial suspension

Biofilms

Ia

II

III

IV

II/IV

II/I

IV/I

I

II

III

IV

II/IV

II/I

IV/I

251.8

469.4

644.8

784.3

0.6

1.9

3.1

171.2

148.7

419.7

307.4

0.5

0.9

1.8

0.6

316.1

587.2

672.3

830.8

0.7

1.9

2.6

150.3

144.3

321.6

228.1

0.6

1.0

1.5

1.2

159.0

538.3

532.8

846.8

0.6

3.4

5.3

100.5

101.1

321.6

284.2

0.4

1.0

2.8

2.4

165.4

584.0

583.0

912.8

0.6

3.5

5.5

99.5

88.7

308.4

228.1

0.4

0.9

2.3

humic-like matter; II, protein-like matter; III, fulvic acid-like substances; IV, tyrosine like proteins

644 645

33



646

TOC/Abstract Art

647 648 649

34


Page 34 of 34

Physiological Responses of Salinity-Stressed Vibrio sp. and the Effect

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